R. Choudhury et al. / Tetrahedron Letters xxx (2018) xxx–xxx
3
Fig. 2. (a) Absorption spectra of 1 with added ethylene glycol in methanol. (b) Emission spectra of 1 with added ethylene glycol in methanol. (c) Absorption spectra of 1 with
added glycerol in methanol. (b) Emission spectra of 1 with added glycerol in methanol. [1] = 1.0 Â 10À5 M; Inset: Appearance of bright red fluorescence emission in glycerol/
water (9:1) mixture.
dipole moment is generated in the ground state, which becomes
very large upon excitation of an electron in the excited state.
Therefore, any excited state phenomenon including radiative decay
energy and the concomitant quantum yield is very sensitive to the
surrounding of the fluorophore, specifically to the polarity of the
solvents [26]. Moreover, in hydrogen bonding solvents such as in
water excited state energy can dissipate via other non-radiative
Next, we sought to exploit this property of the ICT fluorophores
for quantitative detection of Human Serum Albumin (HSA) in
physiological pH. In a solution of fluorophore 1 (phosphate buffer,
pH 7.4), HSA was gradually added and absorption and emission
spectra were recorded at room temperature. Absorption maxima
of 1 shifted both bathochromically and hyperchromically as more
HSA was added. Emission intensity increased as well with increas-
ing amount of HSA (Fig. 3a). Quantum yield of 1 jumped to 0.34
from less than 0.01 in presence of 1% of HSA. All the changes in
spectral characteristic indicate successful inclusion of 1 within
the protein’s cavity. To get a quantitative estimate of the extent
of binding, association constant (Ka) was calculated using
Benesi-Hildebrand relation [31]. As shown in Fig. 3b, plot of
1/(I À I0) vs 1/[HSA] resulted a straight line, indicating 1:1 com-
plexation between 1 and HSA. From the ratio of the slope and
decay channels [29,30]. Therefore, quantum yield of the D—p—A
fluorophores in water is usually low. The quantum yield (UF) of 1
was <0.01 in water with respect to the standard rhodamine B.
Quantum yield of ICT fluorophores comprising flexible or rotat-
able bonds is highly sensitive to the rigidity of the microenviron-
ment. Usually, UF increases as the viscosity of the medium
increases due to suppression of the intramolecular twists of the
rotatable bonds in the main p-conjugated backbone on the excited
state manifold. Therefore, in a high viscous environment, loss of
fluorescence energy via non-radiative pathway is suppressed, and
hence a significant increase in quantum yield is observed [29].
Similar trends were observed for 1 in high viscous liquids such
as ethylene glycol (35.7 cP at 25 °C) and glycerol (950 cP at
25 °C). Fig. 3 shows absorption and emission spectra of 1 in ethy-
lene glycol/methanol and glycerol/methanol mixtures. Absorption
maxima bathochromically shifted 4 nm and 10 nm in 1:1 ethylene
glycol/methanol and glycerol/methanol mixture, respectively.
Emission intensity significantly increased upon addition of ethy-
lene glycol or glycerol in methanolic solution of 1. Emission quan-
tum yields in ethylene glycol/methanol (1:1) and glycerol/
methanol (1:1) were 0.04 and 0.11, respectively. This confirms that
the fluorescence of 1 is highly sensitive to the microenvironment
and has the potential for biosensing applications, since emission
signals can be intensified by restricting the movements and
rotations of the fluorophore within the microenvironment of the
biomolecules.
the intercept corresponding binding affinity (Ka = 1.47 Â 105 MÀ1
)
was calculated.
The high association constant and the corresponding large fluo-
rescence enhancement suggests that in presence of 1 a very small
amount of HSA detection would be possible in aqueous samples. To
determine the detection limit, fluorescence titration of 1 with HSA
was carried out by adding aliquots of HSA in a buffered solution of
1. Fluorescence intensity was plotted as a function of concentration
of HSA and detection limit was calculated from the IUPAC
recommendation of 3r/k equation (Fig. 3c) [32]. It was found to
be 2 mg/L (2 ppm), which indicates that in presence of 1 minimum
amount of HSA can be detected fluorimetrically is 2 mg per liter of
sample. Moreover, fluorescence intensity of 1 at emission maxima
showed an excellent correlation (R2 = 0.991) to the amount of HSA
over the range of 0.020 mg/L to 0.4 mg/L (Fig. 3c). This finding
demonstrates that fluorophore 1 has potential for quantitative
detection of HSA in human urine samples. In this aspect it is impor-
tant to highlight that 1 has reached to the very low detection limit