Dual emission from (E)-3-(4-methylamino-phenyl)-acrylic acid ethyl ester (MAPAEE) and its application as fluorescence probe for studying micellar and protein microenvironment

Article abstract of DOI:10.1016/j.molstruc.2008.07.007

Steady state absorption, fluorescence and time resolved fluorescence spectroscopy have been used for studying the photoinduced intramolecular charge transfer reaction in (E)-3-(4-methylamino-phenyl)-acrylic acid ethyl ester (MAPAEE). The title molecule shows dual emission due to high energy local and a low energy charge transfer emission. Depending on the nature of solvent the red shifted emission band shows good correlation with the solvent polarity parameter, E_T(30) parameter and hydrogen bonding parameter. Quantum chemicals calculations by density functional theory predict that a stabilized twisted intramolecular charge transfer excited state generated either by twisting of the donor group ({single bond}NHMe) or the acceptor (- = {single bond}COOEt) group is responsible for the red shifted charge transfer emission. The solvent polarity dependent red shifted emission from the excited charge transfer state of MAPAEE has been used as fluorosensor to study bovine serum albumin proteinous and sodium dodecyl sulphate micellar microenvironment.

Full text of DOI:10.1016/j.molstruc.2008.07.007

Journal of Molecular Structure 917 (2009) 148–157
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Dual emission from (E)-3-(4-methylamino-phenyl)-acrylic acid ethyl ester
(MAPAEE) and its application as fluorescence probe for studying micellar
and protein microenvironment
Amrita Chakraborty a, Shalini Ghosh a, Samiran Kar a,1, D.N. Nath b, Nikhil Guchhait a,
*
a Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India
b Department of Physical Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata 700 032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Steady state absorption, fluorescence and time resolved fluorescence spectroscopy have been used for
studying the photoinduced intramolecular charge transfer reaction in (E)-3-(4-methylamino-phenyl)-
acrylic acid ethyl ester (MAPAEE). The title molecule shows dual emission due to high energy local
and a low energy charge transfer emission. Depending on the nature of solvent the red shifted emission
band shows good correlation with the solvent polarity parameter, ET(30) parameter and hydrogen bond-
ing parameter. Quantum chemicals calculations by density functional theory predict that a stabilized
twisted intramolecular charge transfer excited state generated either by twisting of the donor group
(ANHMe) or the acceptor (- = ACOOEt) group is responsible for the red shifted charge transfer emission.
The solvent polarity dependent red shifted emission from the excited charge transfer state of MAPAEE has
been used as fluorosensor to study bovine serum albumin proteinous and sodium dodecyl sulphate
micellar microenvironment.
Received 24 April 2008
Received in revised form 7 July 2008
Accepted 10 July 2008
Available online 19 July 2008
Keywords:
(E)-3-(4-Methylamino-phenyl)-acrylic acid
ethyl ester
Dual emission
Twisted intramolecular charge transfer
TDDFT
Bovine serum albumin
Sodium dodecyl sulphate
Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction
In this paper, we have demonstrated the photophysical proper-
ties of (E)-3-(4-methylamino-phenyl)-acrylic acid ethyl ester
Intramolecular charge transfer reactions in donor–acceptor
substituted aromatic systems have been studied vastly by different
groups due to its wide applications. In this process, the charge dis-
tribution takes place between the donor and the acceptor group by
photoexcitation which leads to an increase of the dipole moment
and as well as to structural and electronic rearrangements within
the molecules. Lippert et al. [1] first reported the dual fluorescence
of the benchmark molecule N,N-dimethylamino benzonitrile
(DMABN) and assigned the higher energy emission band to normal
emission of benzene like derivative and the ‘anomalous’, long
wavelength, solvent dependent emission to highly polar, charge
transfer (CT) emission. There are various proposed models, e.g.,
twisted intramolecular charge transfer (TICT), planar intramolecu-
lar charge transfer (PICT), wagged intramolecular charge transfer
(WICT) and rehybridisation intramolecular charge transfer (RICT)
for explaining this dual fluorescence phenomenon of DMABN [2–
5], but the TICT model pioneered by Grabowski and coworkers
[2] has received wide spread attention.
(Scheme 1) in different polarity solvents. The molecule MAPAEE
contains a secondary amino group as charge donor (ANHMe) and
ethyl ester (ACOOEt) as charge acceptor with an extra p-conjuga-
tion between the benzene ring and the acceptor group. The studies
of secondary amino group as charge donor for photoinduced intra-
molecular charge transfer reaction are very uncommon [6–8]. It is
found that secondary amino donor group mainly shows CT emis-
sion when there is an ortho substitution in the ring of the chromo-
phore [2]. Recently, Zachariasse et al. reported similar type of dual
fluorescence for photoinduced intramolecular charge transfer (ICT)
reaction of some secondary amino donor aromatics where fluorine
atoms are substituted in the aromatic ring [8]. Very recently, we
have reported photophysical phenomenon of some aromatic com-
pounds with flexible double bond at the acceptor site and second-
ary amino group without ortho substitution at the donor site [9–
11].We have earlier reported dual fluorescence phenomena in
some self designed donor–acceptor aromatic systems where sec-
ondary amino group without ortho substitution was used as charge
donor with variety of acceptor groups such as nitrile, aldehyde,
acid, methyl ester groups. In the present study, the donor is a sec-
ondary amine and the acceptor is an ethyl ester. We found that the
change of ester group from methyl to ethyl hardly influences the
excited state CT reaction. We have studied photophysical proper-
* Corresponding author. Tel.: +91 33 2432 4159; fax: +91 33 2351 9755.
E-mail address: nguchhait@yahoo.com (N. Guchhait).
Present address: CHEMGEN Pharma International, Dr. Siemens Street, Block GP,
1
Sect. V, Salt Lake City, Kolkata 700 091, India.
0022-2860/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2008.07.007

A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
149
The time resolved fluorescence measurements have been done
with a time correlated single photon counting set up. The exciting
source is frequency tripled output of mode locked picosecond Ti-
Sapphire Tsunami (Spectra Physics, USA) laser pumped by a diode
pumped CW Nd-Vanadate laser (Millenia X, Spectra Physics, USA).
For our measurement the excitation wavelength is 315 nm. A pulse
repetition rate of 82 MHz has been reduced to a repetition rate of
800 kHz/4 MHz by a pulse selector. The average power is in be-
tween 2 and 50 mW. The instrument response function (IRF) is
20 ps. The time interval between the exciting photon (laser pulse)
and a single fluorescence photon from the fluorescent sample ex-
cited by the same laser pulse has been measured in repeated cycles
and the data has been used to construct fluorescence decay of the
sample. The 345 nm filter has been used for removing the scatter
light. The instrument response function (IRF) has been measured
by using a scattering solution of milk powder in water.
All calculations have been performed using Gaussian 03 pack-
age [19]. The geometries of the ground state lowest energy con-
formers have been calculated with B3LYP hybrid functional and
6-31++G(d,p) basis set at DFT level. Time dependent density func-
tional theory (TDDFT) has been used to calculate excitation ener-
gies using the same functional and basis set. In spite of certain
drawbacks [14,15,21], there are various examples where TDDFT
has been used successfully to explore photophysical phenomena
for the ground and excited states [9–12,16–18,20]. Computed exci-
tation energies are the vertical transition energy without zero
point correction. We also extended our calculations in solvated
system using non-equilibrium TDDFT–PCM model [17]. Calcula-
tions on potential energy surface (PES) have been pursued along
the twist coordinates separately at the donor and acceptor sites.
We have used rotational angles h1 and h2 (shown in Scheme 1) to
get the twisting of the donor (ANHMe) and acceptor
(ACH@CHCOOEt) group, respectively. One limitation is that the
above-calculated PES only corresponds to the cut of PE-hyper sur-
face along the twisting angle as no geometry optimization of the
various excited states has been performed. However, this ap-
proach, i.e., the use of ground state optimized geometry as a basis
for the representation of the excited state structure, has been suc-
cessfully applied in many recent scientific publications [16–18,22].
The difference in energy between the S0 and S1 or S2 states of the
calculated PESs without optimization of the excited states at differ-
ent twisted geometry is considered as the emission energy [17].
This value is just an estimation of the emission energy but not a
theoretical estimation of vertical emission from the excited state
since the excited state optimization has not been considered.
H
(-0.2773)
OC2H5
(-0.3369)
N
θ1
θ2
CH3
O
(-0.4892)
Scheme 1. Structure of (E)-3-(4-metylamino-phenyl)-acrylic acid ethyl ester.
Calculated Mullikan charge given in parentheses.
ties of MAPAEE by varying polarity and hydrogen bonding ability of
the solvent, pH and temperature of the medium. Theoretical poten-
tial energy surfaces at density functional theory (DFT) level for the
lower singlet states of MAPAEE have been evaluated as a function
of twisting coordinate along both the donor and acceptor sides
[12]. The effects of solvent on spectral properties have been ex-
plored using polarizable continuum model (PCM) included in the
Time dependent density functional theory (TDDFT) method [13].
Systems demonstrating ICT are recently being widely used as fluo-
rosensors of microenvironment in proteins and micelles. Since the
photophysical properties of MAPAEE were found to be dependant
on the solvent polarities, an effort has also been made to study
the interaction of MAPAEE with protein bovine serum albumin
(BSA) and surfactant sodium dodecyl sulphate (SDS) for the study
of proteinous and micellar microenvironments.
2. Experimental
For the synthesis of MAPAEE, p-nitro benzaldehyde and ethyl
(triphenyl phosphanylidine)acetate (Ph3P@CHACOOEt) in dry
dichloromethane were stirred at room temperature for 24 h. Gen-
erated nitro product was reduced by zinc and saturated aqueous
ammonium chloride solution in methanol at 50 °C for 30 minutes
to produce (E)-3-(4-amino-phenyl)-acrylic acid ethyl ester. Then
one hydrogen atom of amino group was blocked by di-tert-butyl
pyrocarbonate (BOC-anhydride) in presence of triethylamine in
THF to produce (E)-3-(4-amino-phenyl)-acrylic acid. In the next
step, hydrogen atom of BOC protected amino group was replaced
by methyl group using methyliodide in presence of sodium hydride
base in DMF solvent at 0 °C. In the next step, the BOC group was
removed by using trifluoroacetic acid in dichloromethane to pro-
duce (E)-3-(4-metylamino-phenyl)-acrylic acid ethyl ester. The fi-
nal product was purified by column chromatography and
repeated crystallization. 1H NMR (300 MHz, CDCl3) d 1.29 (t, 3H),
2.86 (s, 3H), 4.19 (q, 2H), 6.18 (d, J = 11.85 Hz, 1H), 6.54 (d,
J = 6.51 Hz, 2H), 7.35 (d, J = 6.48 Hz, 2H), 7.57 (d, J = 11.91 Hz, 1H).
The absorption and emission spectra of MAPAEE in different
solvents have been measured by Hitachi UV/VIS U-3501 spectro-
photometer and Perkin-Elmer LS50B fluorimeter, respectively. Sol-
vents used for all measurements are spectral grade from
Spectrochem and E-Mark. The protein BSA and surfactant SDS from
SRL, India, were used for the preparation of proteinous and micellar
solution. All solutions are made at ꢀ10ꢁ6 M concentration of solute
in order to avoid aggregation and self-quenching.
3. Results and discussion
3.1. Absorption spectra
The absorption spectra of MAPAEE in a series of polar, non-polar
and protic solvents have been measured and the positions of the
band maxima are presented in Table 1. The absorption spectra in
non-polar and polar aprotic solvents show a broad band consisting
Table 1
Absorption and emission spectral data of MAPAEE in different solvents at room temperature
Solvent
kabs (nm)
kflu (nm)
mabs (cmꢁ1
)
mflu (cmꢁ1
)
Dm )
(cmꢁ1
Hexane
Dioxane
333, 318
350, 318
351, 318
352, 318
353, 318
358, 318
359, 318
346, 318
382
413
418
422
435
443
454
467
30,030
28,571
28,490
28,409
28,328
27,932
27,855
28,901
26,178
24,213
23,923
23,696
22,988
22,573
22,026
21,413
3852
4358
4567
4712
5501
5359
5828
7488
Chloroform
Tetrahydrofuran
Acetonitrile
Ethanol
Methanol
Water

150
A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
of two overlapping bands, a strong band at ꢀ350 nm and a weak
shoulder one at ꢀ318 nm (Fig. 1a). The spectral pattern is similar
to that of (E)-3-(4-methylamino-phenyl)-acrylic acid methyl ester
(MAPAME), a methyl ester of the same molecule [13]. The conju-
gated double bond and a secondary amino group attached to the
O
H
H
H
H
OEt
H
N
H
O
CH3
benzene ring, results in the red shift of the strong
p–
p* band to
H
O
350 nm due to resonance stabilization. It is found that the peak po-
sition of the low energy band varies slightly with solvent polarity;
but the position of the high energy band is independent of solvent
polarity. Comparing with methyl analogue MAPAME and other
similar studied systems these two absorption bands are assigned
to transition to La(S2) and Lb(S1) state of benzene moiety
[11,12,23]. In protic solvent like water the strong absorption band
(346 nm) is slightly blue shifted and the shifting is such that the
two bands almost overlap to produce a single absorption peak.
As was observed for similar donor–acceptor systems, this blue
shifted band corresponds to the hydrated clusters of MAPAEE [9–
11,24–26]. In the ground state protic solvents act as proton donor
and can form the possible hydrogen bonded clusters (Scheme 2). It
is found that specific hydrogen bonding is an important factor to
favour the ICT process by maintaining a large twist angle between
the electron donor and acceptor group in the ground state [25].
Stabilization of ground state through hydrogen bonding can shift
the absorption band to the blue side.
O
Scheme 2. Structure of hydrated cluster of (E)-3-(4-metylamino-phenyl)-acrylic
acid ethyl ester.
methanol shifts more towards blue side from ꢀ347 nm to
ꢀ270 nm. Obviously the H+ ion of acid prefers to bind to electro-
negative atoms to form a protonated species which absorbs at
ꢀ270 nm. The possible protonation sites are two oxygen atoms
and one nitrogen atom and the calculated negative charge density
at nitrogen atom and two oxygen atoms for the lowest energy
structure support this prediction (Scheme 1). The
p–
p* type of
absorption of such protonated species is similar to that of benzene
chromophore having only a conjugated double bond and therefore,
this protonated species should absorbed at the blue side due to less
resonance stabilization compared to the neutral species.
The absorption spectra in presence of acid are shown in Fig. 1b.
With increase of acidity of the medium the absorption band in
3.2. Emission spectra
Fig. 2 shows the emission spectra of MAPAEE in some selective
solvents when excited at the absorption band maxima. The emis-
sion peaks in different solvents are presented in Table 1. It is found
that the emission spectra MAPAEE are similar to the emission spec-
tra of its methyl analogue MAPAME [11]. As seen in Fig. 2a, a strong
red shifted fluorescence band is observed with increasing polarity
and hydrogen bonding ability of the solvents. Comparing with
other similar reported molecules [9–11] we have assigned the
large red shifted emission in polar aprotic solvent to emission from
the CT state and higher energy emission band to local emission. In
polar solvents the higher energy emission band is broad enough
with high base line value and considered to be the emission from
a locally excited state of the molecule. The lower energy, Stokes
shifted emission band may arise from CT state generated from
the hydrogen bonded clusters in protic solvent [26]. It is reported
that intermolecular hydrogen bonding effect is an interesting sub-
ject to determine proton coupled charge transfer process which is
observed in biological assembles including PS II systems [27]. As
seen in Fig. 2b, the addition of little amount of ethanol in methyl-
cyclohexane solution of MAPAEE shows the generation of new
fluorescence band at the red side with concomitant decrease of ini-
tial emission band observed in pure methylcyclohexane. A clear
isoemissive point at ꢀ398 nm characterizes the excited state
hydrogen bonding complex formation of simple stoichiometry
with protic solvents [25,28]. As was seen in case of methyl ana-
logue the excitation spectra of MAPAEE (figure not shown) of both
the emission bands are independent of emission wavelength and
agree reasonably well with the absorption spectra indicates that
only a single species is present in the ground state and hence the
CT state generated from the LE state.
a
Acetonitrile
Hexane
Methanol
Water
0.1
0.0
300
350
400
Wavelength (nm)
+
b
MeOH+H
MeOH
0.12
0.10
0.08
0.06
0.04
0.02
0.00
As was observed in the absorption spectra, the emission in
methanol is also found to be pH dependent (Fig. 2c). With increas-
ing the acidity of the medium, the intensity of charge transfer (CT)
emission band decreases with increase of intensity of a locally ex-
cited (LE) state emission band of the possible protonated species.
As the proton prefers to bind to the nitrogen lone pair, the lone pair
of nitrogen is now not available for intramolecular charge transfer
reaction; hence the CT band intensity decreases with increasing
acid concentration. Surprisingly, this charge transfer band does
280
350
420
Wavelength (nm)
Fig. 1. Absorption spectra of MAPAEE in (a) different solvents, (b) in presence of
acid (arrow indicates increase of acid concentration).

A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
151
medium. The effect of acid on the absorption and emission spectra
predicts that the CT state is generated from the transfer of charge
from the lone pair of nitrogen atom. From the absorption and emis-
sion spectra of MAPAEE, it is observed that the overall spectral
characteristics of MAPAEE are similar to that of MAPAME. Hence,
the change of substituents of the acceptor ester group does not af-
fect spectral characteristics at all.
To determine the excited state dipole moment of MAPAEE we
have used Lippert plot from the data obtained from absorption
and emission spectra [30].
a
b
c
Acetonitrile
Methanol
1400000
700000
Hexane
Water
ꢀ
ꢁ
2
hca3
ꢀ
ma
ꢁ
mf
¼
D
f½lꢂ
ꢁ
ꢁ
lꢃ2 þ cons tan t
ꢁ
ꢀ
ꢁ
e ꢁ 1
n2 ꢁ 1
where;
D
f ¼
2e þ 1
2n2 þ 1
400
480
In the above equation h, c, a,
l
and l* are the Planck’s constant,
Wavelength (nm)
velocity of light, radius of the cavity in which the fluorophore re-
sides and ground and excited CT state dipole moments, respec-
MCH
tively. The term
and n are the dielectric constant and refractive index, respectively,
of the medium. The ground state dipole moment ( ) and ‘a’ values
Df is known as solvent polarity parameter and e
200
100
0
MCH+15% Ethanol
l
were calculated using hybrid functional B3LYP and 6-31++G(d,p)
basis set for the ground state minimum energy structure and the
values are 5.39 D and 4.5 Å, respectively. Fig. 3a shows a linear
dependency of Stokes shift on solvent polarity parameter. High di-
pole moment of excited CT state (9.8 D) calculated by solvatochro-
mic method again confirms the charge transfer nature of the excited
state.
The large deviation from linearity in Lippert plot in case of prot-
ic solvent indicates that hydrogen bonding may play a key role in
ICT path. As seen in Fig. 3b, the correlation of Stokes shift with sol-
vent dependent properties ET(30) parameter [31] follows two dis-
tinct lines, one for the non-protic solvents and the other for the
protic solvents. This clearly indicates the presence hydrogen bond-
ing interaction along with the dipolar interaction in polar protic
solvents. A linear correlation between the lower energy emission
350
400
450
500
Wavelength (nm)
MeOH
band maximum and hydrogen bonding parameter (a) [32] of protic
+
MeOH+H
solvents again supports the fact (Fig. 3c) that hydrogen bonding
causes preferential solvation of ICT state by lowering its energy va-
lue and facilitate CT process in the excited state. The measure-
ments of luminescence quantum yield and excited state
depopulation kinetics data are collected in Table 2. With the help
of excited state lifetimes and quantum yields it is possible to calcu-
late both the radiative decay rate constant (kf) and the sum of the
rate constants of parallel non-radiative processes (knr). The values
of kf and knr are calculated by the following equations [28].
400
200
kf ¼ /f =sf and knr ¼ ð1=sf Þ ꢁ kf
Fluorescence quantum yields are quenched by protic solvents com-
pared to polar aprotic solvents. In protic solvents the quantum
0
yields value decrease with increasing
a parameters, describing the
350
400
450
500
550
acidity of the hydrogen bonding solvents. This indicates the exis-
tence of non-radiative decay channels of increasing importance
with growing proton donating ability of the solvent. In hexane, MA-
PAEE shows a very short fluorescence lifetime value of ꢀ12 ps, even
lower than the IRF (20 ps). The high non-radiative rate in hexane
indicates that additional radiationless channels may be present in
non-polar solvent as was observed by Maus et al. [33]. With
increasing the polarity of the medium, i.e., in acetonitrile solvent
the lifetime value increases to 27 ps with a negative component
of small contribution. We are not sure about this growth. The higher
radiative rate in acetonitrile with respect to hexane can be ex-
plained by considering the fact that with increasing the polarity
of the medium the CT state become more stabilized leading to less
Wavelength (nm)
Fig. 2. Emission spectra of MAPAEE in (a) different polarity solvents,
(kext = 340 nm), (b) in methylcyclohexane + ethanol mixed solvent and (c) in
presence of acid (arrow indicates increase of acid concentration) (kext = 270 nm).
not totally disappear even in high acid concentration and both the
LE of the protonated species and CT band coexist. This can be pos-
sible if the LE state of the protonated species can undergo excited
state deprotonation reaction to some extent [29]. The deproto-
nated excited state immediately transforms to the CT state in the
excited state potential energy surface, hence both the emission of
the protonated species and the CT state appears in highly acidic

152
A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
The emission of MAPAEE in ethanol glass matrix is shown in
a
Fig. 4. The emission band maximum at 77 K is found to be largely
blue shifted. This may reflect the change in solvent properties such
as polarity, polarizability and viscosity upon decreasing the tem-
perature. The high viscosity of the 77 K glass matrix also inhibits
the relaxation process from LE to CT state through any expected
twisting path. Hence only LE emission should be observed in low
temperature glass matrix. This observation may support the pro-
posed mechanism of TICT where twisting of either the donor or
acceptor side are responsible for red shifted emission band
[34,9,10].
Acetonitrile
5500
5000
4500
4000
DMF
Tetrahydrofuran
Chloroform
Dioxane
CCL4
3.3. Theoretical calculation
To get a better insight into the excited state processes we have
performed structural calculations and evaluated PESs in the light of
TICT model using Gaussian 03 software. The ground state lowest
energy optimized structure has been calculated at DFT level with
B3LYP functional and 6-31++G(d,p) basis set and selective param-
eters are presented in Table 3. In the optimized geometry both the
donor and acceptor groups are found to be planar with the benzene
ring and are similar to its methyl ester compound MAPAME [11].
However in most of the reported donor–acceptor charge transfer
systems the nitrogen group has been found to be twisted with re-
spect to the benzene ring. Considering the TICT model, the posi-
tions of the computed absorption and emission bands for the
ground state global minimum structure are shown in Table 4.
The steady state absorption spectra of MAPAEE in hexane solvent
appears with a strong band maximum at 333 nm (3.72 eV) and
the weak one at 318 nm (3.89 eV) and the same band in acetoni-
trile at 351 nm (3.53 eV) and 318 nm (3.89 eV). The calculated ver-
tical transition energies to the S1 and S2 states in vacuum at TDDFT/
B3LYP/6-31++G(d,p) level (we may consider that the calculations
in vacuum is almost equivalent to that in non-polar solvent) are
330 nm (3.75 eV) and 290 nm (4.27 eV), respectively, and that in
acetonitrile solvent using PCM-TDDFT/B3LYP/6-31++G(d,p) meth-
od are 362 nm (3.42 eV) and 292 nm (4.24 eV), respectively. So
the deviation between the calculated transition energy and exper-
imental band position in hexane and in acetonitrile is only 0.03 eV
and 0.11 eV, respectively. Calculations in vacuum and acetonitrile
solution indicate that the absorption band maximum observed in
the experiment corresponds to the S1 state which has the larger
calculated oscillator strength value (f = 0.851 eV), i.e., this transi-
Hexane
0.00 0.05
0.10
0.15
0.20
0.25
0.30
0.35
f
Δ
b
8000
Water
7000
6000
Methanol
Ethanol
ACN
5000
THF
CHCl
Dioxane
3
Isopropanol
4000
3000
Hexane
36
28
32
40
44
48
52
56
60
64
(
ET(30) Kcal/mole)
23000
c
Isopropanol
Ethanol
22500
22000
tion is of
in the ground state support the nature of HOMO–LUMO transition
is of
p* character (Fig. 5a and b). In MAPAEE, it is assumed that
p–p
* character. The molecular orbital picture of MAPAEE
p–
Methanol
two types of twisting can be possible- (i) twisting along the donor
(ANHMe) group (h1), (ii) twisting along the acceptor (- = ACOOEt)
(h2). For the construction of the energy curves, the geometry is kept
frozen to the optimized ground state at any point and for any elec-
tronic state; the only variable parameter is the twist angle which
has been varied from 0° to 100° with 10° increments. It is clear
from Fig. 6 that along any of the twisting path both in vacuum
and in acetonitrile the ground state shows a single well potential
where orthogonal conformation, either due to twisting of donor
or for twisting of acceptor is the saddle point. Along both the twist-
ing path, both in vacuum and in acetonitrile solvent, the S1 state
shows an asymmetric double well potential, i.e., initially excited
S1 state yields another minimum in the potential energy surfaces
at orthogonally twisted geometrical orientation (Fig. 6). Both the
twisting paths predict the formation of TICT state from LE state
by crossing through a small energy barrier. The calculated barrier
is found to be higher in the gas phase (which is considered to be
same as non-polar solvent) than in acetonitrile solvent. Therefore,
in hexane solvent, the transformation from LE to CT is less favour
and only LE emission is expected in non-polar solvents. On the
21500
Water
0.75
0.80
0.85
0.90
0.95
1.00
1.05
1.10
α
Fig. 3. (a) Plot of Stokes shift vs. solvent parameter (
(b) Plot of Stokes shift vs. ET(30) parameter and (c) plot of fluorescence band
maximum of MAPAEE vs. hydrogen bonding parameter ( ).
Df) (Lippert plot) of MAPAEE,
a
mixing with the forbidden Lb type state. In methanol solvent the
lowering of fluorescence lifetime value and increase of non-radia-
tive rate constant with respect to acetonitrile solvent again support
that hydrogen bonding acts as non-radiative path in ICT reaction.

A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
153
Table 2
Quantum yields and emission lifetime of MAPAEE in selective solvents
Solvent
a
/
s
1(ns)(a1
)
s
2(ns)(a2
)
h
si (ns)
v2
kf ꢄ 10ꢁ9 (sꢁ1
)
knr ꢄ 10ꢁ9 (sꢁ1
)
total
Hexane
Acetonitrile
Ethanol
Methanol
Water
–
–
0.86
0.98
1.10
1.3 ꢄ 10ꢁ2 (LE)
8.23 ꢄ 10ꢁ2 (CT)
4.1 ꢄ 10ꢁ2 (CT)
3.0 ꢄ 10ꢁ2 (CT)
6.4 ꢄ 10ꢁ3 (CT)
0.011(0.376)
0.027(1.00)
–
0.016(0.07)
–
0.013(0.624)
0.012
–
–
0.017
–
3.5
2.0
–
1.0
–
1.08
3.07
–
1.76
–
82.25
33.96
–
57.05
–
0.1(ꢁ0.035)
–
0.018(0.93)
–
Table 4
50
40
30
20
10
Ethanol (77 K)
Computed parameters of MAPAEE in vacuum and acetonitrile solvents using DFT
method with B3LYP hybrid functional and 6-31++g(d,p) basis set
Ethanol (room temperture)
Medium
State
Absorption
Emission
a
b
Eth (eV)
Eex (eV)
Eth (eV)
Eth (eV)
Eex (eV)
Vacuo
S1
S2
3.75
4.27
–
–
3.19
4.20
3.28
4.23
–
–
Acetonitrile
S1
S2
3.42
4.24
3.53
3.89
2.90
4.01
2.94
4.29
2.91
–
Eth is the calculated energy value (Eexcited ꢁ Eground
31++G(d,p)).
) at DFT level (B3LYP/6-
Eex is the experimental value.
a
Emission energy due to twisting of ANHMe group.
Emission energy due to twisting of ACH@CHACOOEt group.
b
0
pbenzene–pacceptor type (f = 0.0). As seen in the molecular-orbital pic-
400
450
Wavelength (nm)
500
550
tures shown in Fig. 5a and b, the nitrogen lone pair is found to be
delocalized over the benzene ring in the HOMO of ground state
optimized structure (Fig. 5a) and acceptor twisted geometry
(Fig. 5e). On the other hand nitrogen lone pair is localized in the
HOMO of donor twisted geometry (Fig. 5c). Therefore, it seems that
delocalization of nitrogen lone pair over benzene ring does not
support CT process along the twisting of acceptor site or in the
un-twisted state. Where as the localized nitrogen lone pair sup-
ports CT process along the twisting of donor group as there are
available lone pair at the nitrogen centre at donor twisted
geometry.
Fig. 4. Emission spectra of MAPAEE at 77 K in ethanol glass matrix and in ethanol
solvent.
Table 3
Optimized geometry for the ground state of MAPAEE in vacuo using DFT (B3LYP/6-
31++g(d,p)) method
Bond
Calculated values (Å)
Angle/dihedral angle
Calculated values (°)
RC1AC2
RC2AC3
RC3AC4
RC1AN7
RN7AH2O
RN7AC9
RC4AC8
RC8AC10
RC10AC11
RC11AO12
RC11AO13
RO13AC14
RC14AC15
1.410
1.391
1.407
1.374
1.006
1.447
1.454
1.351
1.470
1.221
1.361
1.445
1.517
\N7AC1AC2
122.11
117.47
119.32
116.26
107.62
0.00
0.00
0.00
0.00
180.00
\H20AN7AC1
3.4. Study of interaction of MAPAEE with BSA
\C3AC4AC8
\C11AO13AC14
\O13AC14AC15
\C9AN7AC1AC2
\H20AN7AC1AC6
\C3AC4AC8AC10
\O12AC11AO13AC14
\C11AO13AC14AC15
Fig. 7a shows the change of emission characteristics of MAPAEE
in Tris–HCL buffer of pH 7.0 with increasing BSA concentrations. In
Tris–HCl buffer, MAPAEE exhibits the large red shifted ICT emission
band originating from the polar CT emissive state. As seen in
Fig. 7a, with increase of protein concentration the CT emission
band of MAPAEE shifts to the blue with increase of intensity. The
blue shift of the emission band indicates that the probe binds to
the less polar hydrophobic interior part of protein compared to
that of aqueous phase. A more rigid environment is also encoun-
tered by MAPAEE inside the protein. The non-radiative channels
operative in the aqueous phase are less operative in the hydropho-
bic protein environment and hence the fluorescence intensity of
MAPAEE increases with increasing protein concentration.
A quantitative estimate or the extent of binding of MAPAEE to
BSA is determined using the Benesi–Hildebrand relation [35]. The
binding or complexation equilibrium for the 1:1 complex can be
expressed as
other hand polar solvent can tune this energy barrier and CT emis-
sion is expected through LE state excitation as the barrier is low.
Along both the twisting paths, the calculated transition energy
for the orthogonal twisted conformers is very close to the experi-
mentally observed emission value (Table 4). In acetonitrile solvent
the observed CT emission band is at 435 nm (2.91 eV) and the cal-
culated transition energy due to orthogonal twisting of donor is
427 nm (2.90 eV) and that for twisting of acceptor is 421 nm
(2.94 eV). The calculated oscillator strength values and molecular
orbital picture support total decoupled nature of the interacting
states in both the orthogonal orientations. Due to twisting of
ANHMe group (h1 = 90°), the CT excitation is characterized by n–
p* type transition (f = 0.0) (Fig. 5c and d), where as at the twisting
of acceptor group (h2 = 90°) (Fig. 5e and f), the single excitation is
K
MAPAEE þ BSA $ Complex
Benesi–Hildebrand relation for these types of complexation pro-
cesses is expressed in terms of fluorescence intensity with the
assumption that [BSA] >> [Complex], i.e., concentration of the com-
plex is very low compared to that of the free protein. Hence, free
protein concentration can be replaced by total protein concentra-

154
A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
Fig. 5. Molecular orbital picture (a) HOMO of ground state optimized structure, (b) LUMO of ground state optimized structure, (c) HOMO of 90° twisted conformer at donor
side, (d) LUMO of 90° twisted conformer at donor side, (e) HOMO of 90° twisted conformer at acceptor side, (f) LUMO of 90° twisted conformer at acceptor side.
a
b
S
S
2
2
-671.42
-671.44
-671.40
-671.42
S
1
S
1
-671.46
-671.56
-671.44
-671.54
2.90 eV
3.19 eV
S
0
S
0
3.75 eV
3.42 eV
-671.56
-671.58
0
40
80
0
40
80
Twist angle (Degree)
Twist angle (Degree)
c
- 671.40
- 671.42
- 671.44
- 671. 46
- 671. 48
d
S
2
-6 71.40
-6 71.42
-6 71.44
S
2
S
1
S
1
3.28 eV
2.94 eV
S
0
- 671. 57
S
3.75 eV
0
3.42 eV
-6 71.56
- 671. 58
0
20
40
60
80
100
0
20
40
60
80
100
Twist angle (Degree)
Twist angle (Degree)
Fig. 6. Potential energy surfaces of MAPAEE along the twisting of (a) donor side in vacuum (h1), (b) donor side in acetonitrile (h1), (c) acceptor in vacuum (h2), (d) acceptor side
in acetonitrile (h2).

A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
155
1
1
1
50
40
30
20
10
¼
þ
a
ðI ꢁ I0Þ ðI1 ꢁ I0Þ ðI1 ꢁ I0ÞK½BSAꢃ
where I0, I and I1 are the emission intensities in absence of, interme-
diate and infinite concentration of BSA, respectively. As seen in
Fig. 7b, the plot of 1/[IꢁI0] vs. 1/[BSA] shows a straight line indicat-
ing 1:1 complexation between MAPAEE and BSA. From the ratio of
intercept and slope of Benesi–Hildebrand plot the extent of binding
(K) is determined to be 0.818 ꢄ 104 molꢁ1. The associated free en-
(VI)
(I)
ergy change,
DG, for the binding process was then calculated to
be ꢁ22.32 kJ/molꢁ1. The large binding constant indicates strong
binding of the CT probe to the hydrophobic part of BSA protein
and the free energy change favours spontaneous complexation
process.
The blue shift of the CT band indicates that the micropolarity at
the binding site of protein back bone is different from that of aque-
ous phase. Fluorescent probe techniques have been successfully
used in determining the micropolarity of environments in proteins,
micelles, cyclodextrin cavities etc. [36–41]. The Reichardt ET(30)
scale [31] based on the transition energies of the ICT band of beta-
ine dye in various solvent polarities has been widely used to esti-
mate micropolarities of the environments [36–41]. Here we have
attempted to get an idea about the local polarity of MAPAEE bound
to BSA by using the ET(30) scale. As the position of the CT emission
band of MAPAEE bound to BSA (440 nm) appears in between the
position of the same band in dioxane (413 nm) and water
(467 nm) it can be said that the micropolarity of the protein envi-
ronment should definitely be in between the polarity of pure diox-
ane and pure water. The ICT emission of MAPAEE was monitored in
various water–dioxane mixtures by comparing the position of the
CT band of BSA bound MAPAEE with those in different water–diox-
ane mixtures of known ET(30) values. Fig. 7c shows a clear estimate
of local polarity of the protein at the probe binding hydrophobic
site. From the figure it is seen that the ET(30) is 46.7 and it is in be-
tween water and dioxane.
0
380
400
420
440
460
480
500
520
Wavelength (nm.)
b
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
In the Fluorescence Resonance Energy Transfer (FRET) process a
donor molecule is excited at its specific excitation wavelength and
this molecule by dipolar interaction transfers the energy non-rad-
iatively to an acceptor molecule lying within Förster distance of 2–
8 nm from it. The efficiency of FRET depends on three parameters-
(i) the distance between donor and acceptor must be within the
specified Förster distance of 2–8 nm; (ii) there must be appreciable
overlap between donor fluorescence and acceptor absorption band
and (iii) proper orientation of the transition dipole of the donor and
the acceptor fluorophore. According to Förster, the efficiency (E) of
FRET process can be expressed by the following simple equation:
2000
4000
6000
8000
10000
1/conc. of BSA (M-1
)
c
470
460
450
440
430
420
MAPAEE+BSA
MAPAEE+SDS
F
F0
E ¼ 1 ꢁ
ð1Þ
where E is the efficiency of energy transfer; F and F0 are the fluores-
cence intensities of donor in presence and absence of the acceptor,
respectively.
It is found that the emission curve of tryptophan of BSA overlap
well with the absorption curve of the fluorescence probe MAPAEE.
Therefore we have excited tryptophan at 280 nm and monitoring
the emission of tryptophan (kem = 350 nm) and MAPAEE
(kem = 460 nm). Fig. 8 shows the variation of fluorescence spectra
of tryptophan of BSA with increasing concentration of probe MA-
PAEE. On gradual addition of MAPAEE it is seen that the intensity
of tryptophan emission decreases with the emergence of a new
band at ꢀ460 nm. This implies that the radiation emitted by tryp-
tophan of BSA is transferred to the probe MAPAEE by the process of
FRET leading to an increase in emission intensity of the probe mol-
ecule. The efficiency of energy transfer from tryptophan to the
fluorescence probe is found to be ꢀ93%, i.e., the energy transfer
efficiency from tryptophan to the probe is quite high. High FRET
35
40
45
50
55
60
65
ET-(30)
Fig. 7. (a) Emission of MAPAEE in (I): 0, (II):10, (III): 20, (IV): 50, (V): 60 and (VI):
70
l
M BSA in Tris–HCl buffer (pH 7.0), (b) the Benesi–Hildebrand plot of 1/(IꢁI0) vs.
1/[BSA], (c) calibration curve for micropolarity determination in proteinous and
micellar environment: Plot of emission maxima of MAPAEE in water–dioxane
mixtures against corresponding ET(30) values.
tion. The binding constant relation can be expressed by replacing
the concentration of the components in terms of fluorescence inten-
sity as

156
A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
35
a
250
200
150
100
50
30
25
20
15
10
5
(IX)
(I)
0
300
350
400
450
500
0
400
420
440
460
480
500
520
Wavelength (nm.)
Wavelength (nm)
Fig. 8. Fluorescence spectra of tryptophan of BSA with an increasing concentration
of MAPAEE. Arrow indicates increasing concentration of MAPAEE.
35
b
efficiency also supports the strong nature of binding of BSA with
MAPAEE molecule.
30
25
20
15
10
5
3.5. Interaction of MAPAEE with SDS
ICT probes are being widely used by many workers to investi-
gate interactions and/or local environments of bio-mimicking
micellar aggregates [38,42–44]. The changes of the local environ-
ment that take place when the solvent polarity sensitive probe
moves from the bulk aqueous phase to the micellar region in turn
produce prominent changes in the spectral characteristics of the
probe. We have seen that the red shifted emission maxima of MA-
PAEE has its origin in a polar charge transfer (CT) state. Even slight
changes in polarity of the surrounding medium will then cause
shifting of this ICT emission position which serves as a good way
of to monitor micelle–MAPAEE interactions. Fig. 9a shows the ef-
fect of increasing SDS concentrations on the emission characteris-
tics of MAPAEE. A clear blue shift of this maximum from 467 nm in
water to 458 nm in 12 mM SDS is observed which is also accompa-
nied with a simultaneous increase in emission intensity. Blue shift
of the polarity sensitive ICT emission hints at a lowering of local
polarity as the probe moves from the bulk aqueous phase to the
micellar environment. Here again, we have interpolated the posi-
tion of the emission maxima of MAPAEE in 12 mM SDS on the pre-
viously mentioned water–dioxane calibration curves for
micropolarity determination (Fig. 7c). The polarity of the local
micellar environment was then found to be 56.17 on the ET(30)
scale. This value suggests that in SDS, MAPAEE is no longer resident
in the highly polar aqueous phase (ET(30) = 63.1) but rather
encounters a local environment polarity which is somewhere in
between the polarities of pure methanol (ET(30) = 55.4) and pure
water (ET(30) = 63.1). However, this ET(30) value also suggests that
in the SDS micelles MAPAEE does not encounter a microenviron-
ment similar to non-polar alkanes. Hence, we can say that the
probe does not penetrate deep into the hydrophobic core of the
SDS micelle. Instead, in all probability, MAPAEE lies in the water-
micelle interfacial region where it has been reported that the
polarities of the local environment are usually in between those
of pure water and pure alcohols [38].
2
6
0
4
8
10
12
Conc. of SDS (mM)
Fig. 9. (a) Variation of emission characteristics of MAPAEE with increasing
surfactant (SDS) concentrations: (I) 0, (II) 4, (III) 5, (IV) 6, (V) 7, (VI) 8, (VII) 9,
(VIII) 10 and (IX) 11 mM SDS concentration, (b) plot of intensity of emission
maxima of MAPAEE against SDS concentration.
creases steadily till 8 mM SDS concentration after which the in-
crease becomes much less steep. The distinct break in these two
lines occurs at 8 mM SDS concentration which is the reported
CMC of SDS. Therefore, MAPAEE molecule could be used as a good
fluorosensor for determination of CMC of micelles [43,44].
4. Conclusion
The photophysical properties of MAPAEE have been investi-
gated in different solvent by absorption and emission measure-
ment in combination with DFT calculations and all spectral
findings are similar to that of its methyl ester compound, i.e., the
change of ester group from methyl to ethyl does not change spec-
tral characteristics. The molecule clearly shows excited state intra-
molecular charge transfer reaction characterized by solvent
polarity dependent red shifted emission band. In protic solvent
the molecule forms intermolecular hydrated clusters which facili-
tate ICT process. Theoretical calculations predict the formation of a
stabilized twisted intramolecular charge transfer state by twisting
both the donor and acceptor groups. Although, there is a barrier
along the twisting coordinates, but the emission spectra in pres-
ence of acid and localized lone pair at the donor twisting geometry
Fig. 9b shows the variation CT emission intensity of MAPAEE
with increasing concentrations of SDS. Correlation of ICT emission
intensity with SDS concentrations follows two distinct lines, one
for the surfactant concentrations before and one for the same after
the critical micellar concentration (CMC). The CT emission in-

A. Chakraborty et al. / Journal of Molecular Structure 917 (2009) 148–157
[12] C.J. Jodicke, H.-P. Luthi, J. Am. Chem. Soc. 125 (2003) 252.
157
support that the twisting of donor group may be responsible for
the solvent dependent Stokes shifted emission. The solvent polar-
ity dependent CT emission band is used as fluorosensor to deter-
mine local micropolarity of protein and micellar medium.
Spectral modulation of MAPAEE in presence of BSA well reflects
the nature of binding of probe to protein hydrophobic back bone
and fluorescence resonance energy transfer process. It is also used
as sensor to determine ET(30) polarity parameter and critical
micellar concentration of SDS micellar aggregates.
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