Enhancing excited state intramolecular proton transfer in 2-(2′-hydroxyphenyl)benzimidazole and its nitrogen-substituted analogues by β-cyclodextrin: The effect of nitrogen substitution

Article abstract of DOI:10.1021/jp311438s

Excited state intramolecular proton transfer (ESIPT) in nitrogen-substituted analogues of 2-(2′-hydroxyphenyl)benzimidazole (HPBI), 2-(2′-hydroxyphenyl)-3H-imidazo[4,5-b]pyridine (HPIP-b), and 2-(2′-hydroxyphenyl)-3H-imidazo[4,5-c]pyridine (HPIP-c) have b

Full text of DOI:10.1021/jp311438s

Article
pubs.acs.org/JPCA
Enhancing Excited State Intramolecular Proton Transfer in 2‑(2′-
Hydroxyphenyl)benzimidazole and Its Nitrogen-Substituted
Analogues by β‑Cyclodextrin: The Effect of Nitrogen Substitution#
Francis A. S. Chipem, Santosh Kumar Behera, and G. Krishnamoorthy*
Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India
S
* Supporting Information
ABSTRACT: Excited state intramolecular proton transfer (ESIPT) in nitrogen-
substituted analogues of 2-(2′-hydroxyphenyl)benzimidazole (HPBI), 2-(2′-hydrox-
yphenyl)-3H-imidazo[4,5-b]pyridine (HPIP-b), and 2-(2′-hydroxyphenyl)-3H-imidazo-
[4,5-c]pyridine (HPIP-c) have been investigated in a β-cyclodextrin (β-CD) nanocavity
and compared with that of HPBI. The stoichiometry and the binding constants of the
complexes were determined by tautomer emissions. Both pKa and NMR experiments
were employed to determine the orientation of the molecules inside of the β-CD cavity.
Huge enhancement in the tautomer emission of HPIP-b and HPIP-c compared to that
of HPBI in β-CD suggests that not only is the ESIPT favored inside of the cavity, but
also, the environment reduces the nonradiative decay through the formation of an
intramolecular charge-transfer (ICT) state. Unlike HPBI, the tautomer emission to
normal emission ratio of HPIP-b increases from 0.9 to 2.6, and that of HPIP-c increases
from 4.9 to 7.4 in 15 mM β-CD. The effect of dimethylsulfoxide (DMSO) on
complexation was also investigated for all three guest molecules. In DMSO, HPBI is
present in neutral form, but the nitrogen-substituted analogues are present in both neutral and monoanionic forms. However, in
DMSO upon encapsulation by β-CD, all three molecules are present in both neutral and monoanionic forms in the nanocavity.
The monoanion is stabilized more inside of the β-CD cavity. The studies revealed that the ESIPT of nitrogen-substituted
analogues is more susceptible to the environment than HPBI, and therefore, they are more promising probes.
reviewed by Nau et al.18 Wagner more recently reviewed the
1. INTRODUCTION
hydrogen bonding of excited states in supramolecular inclusion
Studies on organized microheterogeneous assemblies have been
growing rapidly during the past few decades as these serve as
complexes including CDs.19
Douhal et al. investigated the effect of β-CD on the dynamics
good miniature models for studying and mimicking important
phenomena in biosystems.1−3 Among the studies on micro-
of an excited-state intramolecular proton transfer (ESIPT)
molecule.16 Warner et al. studied the dual fluorescence of 10-
heterogeneous media, inclusion complexes are one of the
interesting subjects for many researchers. Inclusion complexes
provide valuable information about noncovalent intermolecular
interactions between the host and guest molecules where the
hydroxybenzo[h]quinone and also 2-(2′-hydroxyphenyl)-
benzimidazole (HPBI), its corresponding benzoxazole, and
benzothiazole in the presence of β-CD.20,21 They showed the
existence of weak intramolecular hydrogen bonding in HPBI
guest component is lying within the cavity of the host molecule
without forming any covalent bond.1 Apart from biomimick-
and formation of strong intermolecular hydrogen bonds with
ing,2,4 inclusion complexes find applications in drug delivery,3
the hydroxyl groups of CD. They further showed that the
nanosized electronic devices,5 and energy storage devices.6
phototautomers exist as zwitterions. More recently, Guchhait et
Cyclodextrins (CDs)1−3,5 are the most sought systems for
al. studied the ESIPT process of 1-hydroxy-2-napthaldehyde in
CDs.22
studying such inclusion complexes. CDs are cyclic oligosac-
charides composed of glucopyranose units linked by α-(1,4)
HPBI and its analogues form a class of fluorophores that are
extensively studied due to the ESIPT exhibited by them.21,23−40
bonds with hydrophilic external walls and an interior
hydrophobic nanosized cavity of different size and shape.1−3
Hence, this class of compounds finds applications as lasers,
probes, sensors, and devices.25,34,41−43 The ESIPT reaction is
greatly affected by the hydrogen bonding capability of the
solvent.44−49 In protic solvents, the intramolecular hydrogen
bonded ring in the fluorophore molecule might break to form
The hydrophobicity of the cavity enhances the solubility7 and
the fluorescence of the encapsulated guest molecule.8 There-
fore, CDs have been used as microenvironments to study
excited-state processes such as proton transfer,8−10 charge
transfer,11−13 and energy transfer.14,15 Douhal et al. reviewed
the dynamics and structural aspects of the host−guest
interaction in CD.16,17 The inclusion complexes of fluorophore
in aqueous host molecules including CDs were recently
Received: November 19, 2012
Revised: April 26, 2013
Published: April 26, 2013
© 2013 American Chemical Society
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an intermolecular hydrogen bond between the fluorophore and
the solvent molecules. This reduces the formation of the
phototautomer, which decreases the tautomer emission and
increases the normal emission. However, in aprotic solvents,
the tautomer emission is predominant, and the normal
emission is weak. In addition, at different pHs, these molecules
exist in different forms, namely, neutrals, anions, cations,
zwitterions, and tautomers, giving different emissions.
127.02, 128.86, 132.84, 144.67, 153.50, 158.47. IR (KBr
pellets): 3432, 3064, 1634, 1588, 1491, 1422, 1259, 740 cm−1.
HPIP-c: 1H NMR (400 MHz, DMSO-d6) δ 6.98 (1H, d, Ar−
H), 7.04 (1H, d, Ar−H), 7.39 (1H, t, Ar−H), 7.69 (1H, d, Ar−
H), 8.11 (1H, d, Ar−H), 8.28 (1H, d, Ar−H), 8.94 (1H, s, Ar−
H). 13C NMR (300 MHz, DMSO-d6) δ 109.85, 113.63, 117.38,
119.57, 127.64, 132.57, 136.89, 137.77, 139.50, 156.95, 158.27.
IR (KBr pellets): 3505, 3044, 1632, 1590, 1491, 1415, 1288,
1259, 754 cm−1.
Despite the fact that the utility of HPBI as a fluorescent
probe was well-established,22,23,50,51 the utility of its nitrogen
analogues 2-(2′-hydroxyphenyl)-3H-imidazo[4,5-b]pyridine
(HPIP-b) and 2-(2′-hydroxyphenyl)-3H-imidazo[4,5-c]-
pyridine (HPIP-c) are not investigated. Chattopadhyay pointed
out that although the ESIPT process is affected by the
microheterogeneous media, it also strongly depends on the
nature of the molecule.52 Interestingly, substitution of the N-
heteroatom in the benzene ring of HPBI reduces the quantum
yield,35−37,53,54 and on the other hand, nitrogen substitution on
the phenolic ring increases the quantum yield.54−56 Theoretical
calculations predicted that the proton-coupled charge-transfer
state may act as the major nonradiative decay channel for the
proton-transferred tautomer.34,53−59 The ESIPT is accompa-
nied by a torsion rotation of the two aromatic rings, which leads
to an intramolecular charge transfer (ICT) state from where the
nonradiative decay occurs. This should increase environmental
sensitivity of the nitrogen-substituted analogues more than that
of HPBI. Therefore, we also explore the efficiency of these
fluorophores as potential fluorescence probes by investigating
the properties of these molecules in CD utilizing the ESIPT
reaction. β-CD is the most common CD, and it has a sufficient
diameter to encapsulate a variety of aromatic guest molecules
firmly and tightly, while other CDs have a too small or too large
cavity to make strong binding.18,60 It can also accommodate
HPBI and its analogues lengthwise without hindrances. HPBI is
already studied in β-CD,21 and phenylimidazopyridine
derivatives, structurally related molecules of HPBI, also have
a higher binding affinity for β-CD.11,61 Therefore, we have
chosen the β-CD nanocavity for the present studies.
The absorbance and steady-state fluorescence spectra were
recorded with a Cary 100 spectrophotometer and Horiba Jobin
Yvon Fluoromax 4 fluorimeter, respectively. The excitation and
emission slit widths for fluorescence measurements were 1 nm.
Quantum yields were measured using quinine sulfate in 1 N
sulfuric acid (Φf = 0.546)62 as the standard. Lifetime
measurements were performed on a time-correlated single-
photon counting (TCSPC)-based Edinburgh instrument Life-
Spec II using PicoQuant’s 308 nm LED and Edinburgh
instrument’s 375 nm laser diode as light sources with full pulse
widths of 635 and 2 ps, respectively, at half-maximum. The
emission slit widths used in the lifetime measurements were 20
nm in the case of the 308 nm LED and 10 nm in the case of the
375 nm laser diode. The concentrations of the fluorophores
were 5 μM in all of the absorption and fluorescence spectral
measurements performed for the neutral form of the
fluorophores. The concentrations were 10 μM for spectral
1
measurements of monocationic forms. H NMR spectra were
obtained from a 400 MHz NMR spectrometer and 13C NMR
spectra were obtained from a 300 MHz NMR spectrometer. All
of the experiments were performed at room temperature (298
3 K).
3. RESULTS AND DISCUSSION
3.1. Absorption Spectra. HPIP-b and HPIP-c have low
solubility in water, but their solubility enhances in the presence
of β-CD. This enhancement in solubility is an indication of
inclusion complex formation by the encapsulation of the
fluorophores in the hydrophobic cavity of β-CD. The effects of
β-CD on the absorption spectra of HPIP-b and HPIP-c in
water are depicted in Figure 1. Though absorbance maxima of
both HPIP-b and HPIP-c are nearly unaffected with the
increase in concentration of β-CD, a small hyperchromic effect
is observed. These increases in absorbance are attributed to the
detergent action of the host molecule β-CD on the guest
molecule, which also confirms the formation of the host−guest
inclusion complex.63,64
The effects of β-CD on the absorption spectra of HPBI and
its analogues in DMSO are presented in Figure 2. The same as
in aqueous solution, with the addition of β-CD, there is
hyperchromic effect in the absorption spectra of HPBI and
HPIP-c (Figure 2a and c). The changes in absorption spectra of
HPIP-b in DMSO are different in the sense that isosbestic
points are observed (Figure 2b). However, the most striking
difference between DMSO and aqueous solution is the
appearance of a weak new band at around 365 nm in HPIP-b
and HPIP-c. The band’s absorption is very little in DMSO but
increases with an increase in concentration of β-CD in DMSO.
In HPBI though, the red-shifted band is absent in the absence
of β-CD in DMSO; it appears in DMSO at higher
concentrations of β-CD.
2. MATERIALS AND METHODS
HPBI, HPIP-b, and HPIP-c were synthesized by the
condensation of salicylic acid with ortho-phenylenediamine,
2,3-diaminopyridine, and 3,4-diaminopyridine, respectively, in
polyphosphoric acid by following the method described
elsewhere.25,35,37 The compounds were recrystallized four
times in methanol before use. β-CD from Sigma Aldrich and
HPLC-grade dimethylsulfoxide (DMSO) from Merck India
were used as received. Millipore water was used for preparing
aqueous solution. The pH of the solutions was adjusted by
addition of small amount of sodium hydroxide and sulfuric acid
solution.
1
HPBI: H NMR (400 MHz, DMSO-d6) δ 7.01 (1H, d, Ar−
H), 7.05 (1H, d, Ar−H), 7.28 (2H, br, Ar−H), 7.38 (1H, m,
Ar−H), 7.61 (1H, br, Ar−H), 7.70 (1H, br, Ar−H), 8.02 (1H,
d, ArN-H). 13C NMR (300 MHz, DMSO-d6) δ 112.74, 117.38,
118.17, 119.39, 122.67, 123.52, 126.36, 131.99, 151.83, 158.11.
IR (KBr pellets): 3325, 3056, 2508, 1602, 1590, 1492, 1395,
1262, 726 cm−1.
HPIP-b: 1H NMR (400 MHz, DMSO-d6) δ 7.02 (2H, d, Ar−
H), 7.30 (1H, m, Ar−H), 7.42 (1H, t, Ar−H), 8.04 (1H, d, Ar−
H), 8.11 (1H, d, Ar−H), 8.39 (1H, d, Ar−H). 13C NMR (300
MHz, DMSO-d6) δ 112.39, 116.05, 117.63, 119.01, 119.69,
3.2. Fluorescence Emission Spectra. In aqueous solution
upon excitation at 310 nm, HPIP-b exhibits both normal and
tautomer emissions (Figure 3a). A small enhancement is
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Figure 1. Absorption spectra of (a) HPIP-b and (b) HPIP-c at
different concentrations of β-CD in water.
observed in the intensity of the normal emission. However,
there is a significant change in the tautomer fluorescence of
HPIP-b in aqueous solution with increasing β-CD concen-
tration. While the emission maximum of the normal band at
365 nm is nearly unaffected, that of the tautomer band is red-
shifted by 16 nm from 468 nm in an aqueous medium to 484
nm with 15 mM β-CD, with substantial enhancement in
fluorescence. While the full width at half-maximum (fwhm) of
the normal band is 4610 cm−1 in the absence of β-CD, that in
the presence of β-CD is 4155 cm−1. The fwhm of the tautomer
band decreases from 5635 cm−1 in the absence of β-CD to
3170 cm−1 with 15 mM β-CD. The quantum yield ratio of the
tautomer to normal emission (ΦT/ΦN) increases from 0.9 (in
the absence of β-CD) to 2.6 (at a 15 mM concentration of β-
CD; Figure 4). The effects of β-CD on fluorescence
characteristics of HPIP-c in aqueous media are a little different
from that of HPIP-b (Figure 3b). The intensity of the normal
emission is reduced with an increase in tautomer emission with
a quasi-isoemissive point. The presence of a quasi-isoemissive
point is due to the existence of a cis-enol/trans-enol
equilibrium. However, a clear isoemissive point is not observed,
owing to the increase in the radiative decay of trans-enol inside
of the CD cavity. In HPIP-b, the enhancement in radiative
decay of trans-enol upon encapsulation dominates over the
decrease in the population of trans-enol. Therefore, even the
quasi-isoemissive point is not observed (Figure 3a).
Figure 2. Absorption spectra of (a) HPBI, (b) HPIP-b, and (c) HPIP-
c in increasing concentrations of β-CD in DMSO.
versus the dielectric constant of the dioxane−water mixture
(Supporting Information Figure S10), the dielectric constant of
the environment is estimated to be 37 and 34, respectively, for
HPIP-b and HPIP-c. The estimated polarity is in reasonable
agreement with the literature reports that the estimated polarity
of β-CD is similar to that of the methanol−water mixture.11,66
Red shifts are observed in the tautomer bands of HPIP-b and
HPIP-c upon decreasing the polarity of the environment.35,36
Therefore, the red shift suggests the inclusion of the guest
molecule into the hydrophobic cavity of the host β-CD. This is
further substantiated by the decrease in the fwhm of the
tautomer band due to reduction in solvated structures. The
enhancement of tautomer emission in the β-CD complex can
be explained as follows. HPBI and its analogues are present in
both cis- and trans-enol forms.30,35,36,53 cis-Enol is less polar
than trans-enol.53 The closed cis-enol upon excitation
exclusively undergoes ESIPT to form a tautomer, and the
trans-enol upon excitation gives normal emission. Protic solvent
breaks the intramolecular hydrogen bond and forms more
solvated trans-enol that lead to an increase in normal emission.
In HPBI and its nitrogen-substituted analogues, the decreases
in polarity of the environment shifts the cis-enol/trans-enol
equilibrium toward cis-enol.25,35,37 This is evident from the
Compared to that of HPIP-b, the increase in tautomer
emission of HPIP-c is relatively smaller in magnitude. The
tautomer band is red-shifted from 464 nm at 0 mM to 469 nm
at 15 mM β-CD. The fwhm of the tautomer band decreases
from 3715 cm−1 in the absence of β-CD to 3400 cm−1 with 15
mM β-CD. The ΦT/ΦN ratio increases from 4.9 at 0 mM β-CD
to 7.4 at a 15 mM concentration of β-CD (Figure 4). The value
of the effective dielectric constant of the environment was
calculated using a dioxane−water mixture.65 From the plot of
the tautomer emission maxima in a dioxane−water mixture
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equilibrium toward cis-enol, and the increase in the relative
population of cis-enol would result in the enhancement of the
tautomer emission. However, an increase in the relative
population of cis-enol is not the sole factor that leads to an
increase in the tautomer emission. The other factor that is
responsible for the increase in the tautomer fluorescence is the
decrease in the nonradiative decay in the inclusion complex. It
should be noted here that torsional-induced rotational
relaxation of the phototautomer to nonemissive ICT acts as a
nonradiative channel for the deactivation of the excited
tautomer.53,57−59 At twisted geometry, the energy is minimum
in the excited state and maximum in the ground state.53 Thus,
the energy gap is less, which favors the nonradiative
́
deactivation. Rodrıguez et al. found that the radiationless
deactivation channel was enhanced in HPIP-b both upon
deprotonation of the hydroxyl group and protonation of the
pyridine nitrogen.67 The deprotonation of hydroxyl group is
supposed to increase the charge on the donor, and the proton
of the pyridine nitrogen enhances the electron-withdrawing
ability of the acceptor, and therefore, they also attributed the
nonradiative decay to formation of a nonfluorescent ICT
state.67 In a related study, Rodrıguez et al. also reported that
́
when the electron-withdrawing ability of the imidazopydrine
ring of HPIP-b is increased by methylation at the pydridine
nitrogen, it becomes nonfluorescent due to ESIPT followed by
torsional relaxation to the ICT state.39 More recently, Sekiya et
al. detected the twisted conformers of HPBI, those formed
through torsional rotation of the excited tautomer in
polymorphs of HPBI.68 Douhal et al. also reported that the
twisting motion of 2-(2′-hydroxyphenyl)-4-methyloxazole after
ESIPT is restricted by the β-CD nanocavity.69
Figure 3. Fluorescence spectra of (a) HPIP-b and (b) HPIP-c at
different concentrations of β-CD in water (λexc = 310 nm). (* denotes
water Raman)
Warner et al. reported that the fluorescence spectrum of
HPBI in aqueous β-CD remains unaffected except for small red
shift in the longer-wavelength emission.21 However, unlike
HPBI, significant changes are observed in the tautomer
emission of nitrogen-substituted analogues upon encapsulation
in an aqueous β-CD cavity. The tremendous increase is also
observed in the tautomer to normal emission ratio. All of these
suggest that the nitrogen substitution makes HPIP-b and HPIP-
c sensitive to the environment and can be a better ratiometric
probe than HPBI. Our earlier theoretical studies on HPBI and
its analogues suggested that nitrogen substitution increases the
torsional-induced formation of a nonemissive ICT state.53 This
is due to the higher electron-accepting capability of the
imidazopyridyl moiety over the benzimidazolyl moiety. In
addition, hydrogen bonding of the solvent with pyridine
nitrogen increases the electron-withdrawing ability of the
Figure 4. Plot of the quantum yield ratio of the tautomer to normal
band of (a) HPIP-b and (b) HPIP-c against [β-CD] in water (λexc
=
310 nm).
increases in the tautomer to normal band ratios with the
decrease in polarity of the environment. Therefore, upon
encapsulation, the decrease in polarity should shift the
Figure 5. Sites of hydrogen bond formation with the imidazopyridyl moiety of (a) HPIP-b and (b) HPIP-c in a β-CD nanocavity.
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imidazopyridine ring that increases ICT. In 2-(4′-N,N-
dimethylaminophenyl)imidazopyridines also, it is reported
that the hydrogen bonding of the solvent with pyridine
nitrogen induces the ICT.70−72 Rodrıguez et al. found that the
́
pyridine nitrogen methylated derivative of HPIP-b is non-
fluorescent due to the formation of a nonemissive ICT state
after ESIPT.39 The electron-withdrawing ability of the azole
moiety increases with methylation, and hence, the ICT from
the phenolic moiety to the heterocyclic ring is favored more.
Complex formation reduces the interaction of the water
molecule with the pyridine nitrogen, such as reduction in
hydrogen bonding should reduce the electron-withdrawing
ability of the azole moiety. The difference in behavior of HPIP-
b and HPIP-c is due to the position of the pyridine nitrogen
atom. As shown in Figure 5 (see section 3.5 for the
orientation), the hydrogen bonding of the rim hydroxyl
group/water molecule with pyridine nitrogen is less feasible
in the HPIP-b complex than in the HPIP-c complex. The
nonradiative decay increases upon protonation at the pyridine
nitrogen.36,37,67 Therefore, the larger decrease in the non-
radiative decay in HPIP-b than that in HPIP-c may be due to
higher reduction in hydrogen bonding with pyridine nitrogen.
Figure 6 depicts the fluorescence spectra of HPBI, HPIP-b,
and HPIP-c in DMSO at different concentrations of β-CD. As
in an aqueous medium in DMSO, HPBI also exhibits dual
emission due to normal and tautomer fluorescence (Figure 6a).
The intensities of both the bands increase with β-CD
concentration up to 11 mM. With a further increase in β-CD
concentration, the intensity of the tautomer emission increases
with an increase in β-CD concentration. However, that of
normal emission decreases above 11 mM, and a new band starts
to appear at 415 nm. The intensity of the new band increases
with β-CD concentration. On the other hand, in both HPIP-b
and HPIP-c, the additional band appears as a shoulder in
between the normal and tautomer bands even in the absence of
β-CD. With an increase in β-CD, the tautomer emission
undergoes a blue shift with an increase in intensity, and the new
band is buried underneath of the tautomer band. These changes
suggest that in DMSO also, the ESIPT process is favored more
upon formation of an inclusion complex. The normal emission
intensity decreases, and quasi-isoemissive points are observed.
The presence of quasi-isoemissive points indicates the
equilibrium between trans-enol and the anion. The absence of
a quasi-isoemissive point in HPBI (Figure 6a) is due to the
complicated nature of the equilibrium, that is, the absence of an
anion at lower concentration, where the decrease in the
nonradiative decay from trans-enol dominates over other
factors, and the formation of an anion only at higher
concentration.
Figure 6. Fluorescence spectra of (a) HPBI, (b) HPIP-b, and (c)
HPIP-c in DMSO as a function of [β-CD].
following facts: (i) the red shift is observed in the absorption
and the excitation spectra upon deprotonation in HPBI and its
analogues HPIP-b and HPIP-c, and the new band absorption
spectrum is close to that of monoanion absorption spectrum
(see later); (ii) the positions of the bands are consistent with
the fact that the monoanion fluorescence spectrum is red-
shifted with respect to the normal emission and blue-shifted
compared to the tautomer emission.
DMSO acts as proton acceptor, and it can abstract the
proton from a hydrogen bond donor depending on the acidity
of the donor. The resulting anion is stabilized by solvation
(Chart 1). The monoanion is formed in a nitrogen-substituted
analogue even in the absence of β-CD but not in HPBI due to
greater stabilization of the monoanionic form of HPIP-b and
HPIP-c than the monoanionic form of HPBI due to the
presence of a pyridine nitrogen. This was substantiated by the
lower pKa of HPIP-b and HPIP-c than that of HPBI.25,36,37 The
pKa value of HPBI decreases inside of the β-CD due to
stabilization of the monoanion by the β-CD nanocavity;
therefore, addition of β-CD leads to formation of a monoanion
in HPBI.
To confirm the origin of the emitting species, the
fluorescence excitation spectra of all three molecules are
recorded in DMSO at different concentrations of β-CD
(Figures 7−9). The excitation spectra monitored at the normal
emission (Figures 7a, 8a, and 9a) are blue-shifted from the
excitation spectra monitored at the tautomer emission (Figures
7c, 8c, and 9c). This indicates that the normal and tautomer
emissions originate from two distinct ground-state species,
trans- and cis-enols, respectively. The red-shifted absorption
band at around 365 nm is present only in the excitation spectra
monitored at 420 (Figure 7b), 415 (Figure 8b), and 430 nm
(Figure 9b). These show that the new band is due to different
species. It can be assigned to a monoanion formed by the
deprotonation of a phenolic proton. This is supported by the
The formation of the monoanion was further substantiated
by fluorescence decay measurements. The lifetime data of
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Figure 7. Fluorescence excitation spectra of HPBI in DMSO with
increasing concentrations of β-CD monitored at (a) 355, (b) 420, and
(c) 465 nm.
Figure 8. Fluorescence excitation spectra of HPIP-b in DMSO with
increasing concentrations of β-CD monitored at (a) 365, (b) 415, and
(c) 485 nm.
molecule is completely encapsulated in β-CD, respectively, and
K is the association constant.
HPBI, HPIP-b, and HPIP-c in DMSO in the presence and
absence of β-CD are tabulated in Table 1. The lifetimes of the
normal emission of the fluorophores are not affected by the
presence of β-CD. Though the lifetime of the tautomer
emission of HPBI is not affected by β-CD, there is an increase
in the lifetime of the tautomer emission of HPIP-b and HPIP-c.
Using a 375 nm laser diode as the excitation source, the decay
was monitored at 420, 435, and 438 nm for HPBI, HPIP-b, and
HPIP-c, respectively. The lifetime values thus obtained are
different from those of the normal and tautomer emissions.
This confirms the formation of a monoanion.
3.3. Binding Constant. To determine the stoichiometry
and the binding constant of the inclusion complexes, the
fluorescence intensities of the tautomer emissions at different β-
CD concentrations in water as well as DMSO were analyzed
using Benesi−Hildebrand equations73 (eq 1) for 1:1 complex
formation.
The plot of 1/(I − I0) against [β-CD]−1 shows linear
variation, justifying the validity of eq 1 and thus showing the
formation of a 1:1 complex between the fluorophores and β-
CD both in water and in DMSO. The plots of 1/(I − I0)
against [β-CD]−2 could not be fitted linearly (not shown) for
all of the solutions, which rules out the 1:2 complex formation
with β-CD. The association constants obtained from the
Benesi−Hildebrand plot for HPIP-b and HPIP-c with β-CD in
water are 200 and 95 M−1, respectively. Warner and his group
calculated the binding constant for HPBI/β-CD inclusion
complex formation to be 131 M−1 in water.21 In DMSO,
association constants for HPBI, HPIP-b, and HPIP-c are 193,
78, and 70 M−1, respectively.
3.4. pH Titration. The prototropic equilibrium of the
fluorophores is affected by complexation and the calculation of
acidity constants (pKa), which gives useful information about
guest orientation inside of the inclusion complex.21,74,75 Hence,
the acidity constants (pKa) of HPIP-b and HPIP-c in the
presence of β-CD (15 mM) were determined spectrophoto-
metrically with the help of UV−visible absorption measure-
ments at different pHs in aqueous β-CD solutions (Figure 10).
1
1
1
=
+
I − I0
Iα − I0
(Iα − I0)K[CD]
(1)
where I0, I, and I∞ are emission intensities in the absence of β-
CD, at an intermediate β-CD concentration, and when the
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Table 1. Lifetimes of HPBI, HPIP-b, and HPIP-c in the
Absence and Presence of β-CD (15 mM) in DMSO
normal
emission
tautomer
a
a
b
emission
anion emission
χ2
τ1 (ns)
χ2
τ1 (ns)
χ2
N
T
A
sample
HPBI
τ1 (ns)
DMSO
1.5
1.5
1.0
1.0
4.4
4.4
1.0
1.0
DMSO + β-CD
HPIP-b
3.2
1.0
DMSO
1.2
1.2
1.0
1.1
3.2
3.5
1.1
1.2
4.0
4.1
1.1
1.0
DMSO + β-CD
HPIP-c
DMSO
1.2
1.2
1.0
1.0
3.4
3.9
1.1
1.0
3.7
3.9
1.0
1.0
DMSO + β-CD
a
b
λexc = 308 nm. λexc = 375 nm.
Figure 9. Fluorescence excitation spectra of HPIP-c in DMSO with
increasing concentrations of β-CD monitored at (a) 355, (b) 430, and
(c) 465 nm.
Chart 1. Solvated Structures
Figure 10. Absorption spectra of (a) HPIP-b and (b) HPIP-c in 15
mM aqueous β-CD at different pHs.
In the presence of β-CD, HPIP-b is completely in the neutral
form at pH 6.3, while HPIP-c is neutral at pH 5.3. The pKa
value for the neutral−monoanion equilibrium of HPIP-b was
found to be 8.3, which is a little lower than 8.6 in the absence of
β-CD reported by Dogra et al.36 In the case of HPIP-c, also the
pKa value for the neutral−monoanion equilibrium (8.6) is
lower than that in aqueous solution (9.3).37
The fluorophores have two acidic centers, namely, the
phenolic OH and the imidazole NH. Phenol has pKa ≈ 10,
while imidazole has pKa ≈ 14.5. Hence, because the observed
pKa is closer to that of phenol, the monoanion formed is due to
the deprotonation of phenolic OH. The lower pKa value
observed here compared to that of phenol (∼10) may be due to
polarization of the phenolic OH bond as a result of the
presence of an electron-withdrawing imidazopyridine moiety.
Because the pKa of the imidazole >NH group is higher than
that of the hydroxyl proton of the β-CD,76 we did not attempt
to calculate the pKa of the imidazole >NH group.
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A similar decrease in pKa values was observed for 2-(2′-
hydrophenyl)benzazoles21 and other aromatic alcohols.76 The
lowering of the pKa value for the neutral−monoanion in the
presence of β-CD is ascribed to the formation of an
intermolecular hydrogen bond between the phenolic oxygen
and the alcoholic OH groups of β-CD, the alcoholic OH groups
acting as the H-bond donor. This facilitates the stabilization of
the monoanion formed by deprotonation. The difference in the
pKa values in the presence and absence of β-CD is a measure of
the strength of the intramolecular hydrogen bond through
which the ESIPT occurs. It appears that the strength of the
intramolecular hydrogen bond depends on the position of the
pyridine nitrogen and is stronger in HPIP-c than that in HPIP-
b. This was consistent with the fact that ΦT/ΦN is higher in
HPIP-c than that in HPIP-b (Figure 4).
3.5. Orientations of the Guests in Inclusion Com-
plexes and NMR Studies. The length and the interior
diameter of the β-CD cavity are 7.9 and 7.8 Å, respecively.77
The dimensions of the density functional theory (DFT)-
optimized structure HPIP-b using the method B3LYP/6-
31G(d) by Gaussian 03 software78 are shown in Figure 11.53
monoanion clearly suggests that the phenyl ring of the
molecule is present outside of the cavity (Figure 12a). Such
an orientation allows restricted hydrogen bonding with solvent
molecules. It is also feasible for intermolecular H-bonding with
the OH of β-CD.
Formation of the monoanion indicates that HPIP-b and
HPIP-c partially penetrate the β-CD cavity with the
imidazopyridine moiety. The phenolic group resides outside
of the upper rim are exposed to the solvent environment, and
its OH group presents near the rim. HPBI was also reported to
have the same orientation in aqueous β-CD.21
The NMR spectra of the guest in the presence and absence
of β-CD should give a clearer picture of the orientation of the
molecules inside of the β-CD cavity.11 Due to poor solubility,
we are not able to record the NMR in heavy water. However,
1H NMR spectra were recorded in DMSO, and the aromatic
region of the spectra is depicted in Figure 13. NMR decoupling
experiments were performed for proton peak assignments
(Supporting Information). In all three molecules, as expected,
the complexation with β-CD affects protons of the
benzimidazole/pyridoimidazole more than the protons of the
hydroxyphenyl ring. In HPBI (Figure 13a), both Ha and Hb
protons appears as doublets, in the absence of β-CD, but peaks
of both protons are shifted and merged to appear as a broad
singlet at δ 7.653 in the presence of β-CD. Similarly, the Hb and
Hb′ peaks appear as pseudo-triplets at δ 7.286 in the absence of
β-CD, but the splitting disappears to a singlet at 7.277 in the
presence of β-CD. On the other hand, the effect is negligible in
peaks of phenolic ring protons. The chemical shift values for
HPIP-b are in the order He < Hg < Hb < Hf < Ha < Hd < Hc
(Figure 13b). In the absence of β-CD, the Ha proton peak
appears as a doublet at 8.06 ppm, and the Hd proton peak
appears as a doublet at 8.10 ppm. In the presence of β-CD, the
Hd proton is affected less, but Ha proton peaks are shifted
downfield and merged with the Hd proton to appear as a broad
shoulder. The Hc proton appears as a doublet in the absence of
β-CD and merges to appear as a broad single peak in the
presence of β-CD. The well-resolved peaks of the Hb proton in
the absence of β-CD become less resolved in the presence of β-
CD. However, the effect of β-CD complexation is negligible for
protons of the phenolic ring. Similarly, in the case of HPIP-c,
the chemical shift values of Ha are shifted from 8.947 to 8.930,
Hb is shifted from 8.290 to 8.281, and Hc is shifted from 7.687
to 7.680 in the presence of β-CD. As expected, the shift is more
in Ha than that in Hb or Hc. The effect of CD complexation is
also less on phenolic ring protons (Figure 13c).
Figure 11. Optimized geometry of HPIP-b obtained from DFT
calculations.
The dimensions of HPBI and HPIP-c are nearly the same as
those of HPIP-b.53 On the basis of the dimensions of the host
and guest molecules, it can be inferred that the guest can be
encapsulated only partially, and the guest molecule can enter
the cavity of β-CD in two possible ways, as shown schematically
in Figure 12. If the guests were oriented as shown in Figure 12a,
the −OH group of the guest molecules would be present near
the rim. On the other hand, if the guests were present in the
reverse way as shown in Figure 12b, the −OH group of the
guest molecules would be present inside of the cavity. Because
the interior of the CD nanocavity is hydrophobic, the formation
of the monoanion is possible only if the solvent molecule
interacts with guest molecules. The formation of the
Figure 12. Mode of 1:1 complexation of HPIP-b with β-CD with the heterocyclic imidazopyridine moiety (a) inside and (b) outside of the β-CD
nanocavity.
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the imidazopyridine ring. Consequently, monocations are not
encapsulated inside of the hydrophobic CD cavity. Douhal et al.
also demonstrated that ESIPT exhibiting milrinone also enters
the CD cavity through the pyridine ring.80 It appears that
substitution influences the orientation of the molecules.
4. CONCLUSION
HPBI, HPIP-b, and HPIP-c enter the β-CD cavity through the
benzimidazole/imidazopyridine moiety. NMR confirms the
proposed orientation of the molecules. Encapsulation of HPIP-
b and HPIP-c by β-CD enhanced the ESIPT reaction. Though
all three molecules form 1:1 inclusion complexes with β-CD,
the effects of β-CD on HPIP-b and HPIP-c are much more
significant compared to that of HPBI. Remarkable enhance-
ment in the fluorescence and a red shift are observed in the
tautomer emission of HPIP-b and HPIP-c compared to that of
HPBI. The tautomer emission is increased by both an ESIPT-
favorable environment of the β-CD cavity and reduction in the
torsional-induced nonradiative decay through a twisted ICT
state. The pKa for the neutral−monoanion equilibrium of the
molecules decreases due to greater stabilization of the
monoanion inside of the nanocavity. All three molecules
donate the proton to DMSO to form a monoanion in DMSO.
The monoanion of HPBI is formed in DMSO solution above 4
mM β-CD. However, the presence of extra nitrogen stabilizes
the monoanion, and HPIP-b and HPIP-c are present as both a
neutral and monoanion in the presence as well as absence of β-
CD in DMSO. The presence of extra nitrogen makes HPIP-b
and HPIP-c more sensitive to the environment than HPBI, and
therefore, they can be a better environment probe than HPBI.
Unlike the neutral and monoanion, monocations of HPIP-b
and HPIP-c do not form a complex with β-CD.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR, IR spectra of HPBI, HPIP-b, and HPIP-c,
decoupled NMR and assignment of NMR peaks, dielectric
constant determination plots, time-resolved fluorescence
decays, Benesi−Hildebrand plots, NMR titration spectra, and
the fluorescence of monocationic forms at different CD
concentrations. This material is available free of charge via
the Internet at http://pubs.acs.org.
Figure 13. 1H NMR spectra of (a) HPBI, (b) HPIP-b, and (c) HPIP-c
in the presence (solid line) and absence (dotted line) of β-CD in
DMSO-d6.
HPIP-b and HPIP-c monocations are formed by the
protonation of the ring nitrogen.36,37 Therefore, to substantiate
further, the fluorescence characteristics of monocationic forms
of HPIP-b and HPIP-c are measured as a function of β-CD
concentration in aqueous solutions. The fluorescence spectra of
the monocations are unaffected by β-CD (Supporting
Information Figure S18). This suggested that the monocations
are not forming a complex with β-CD. On the other hand,
monocationic forms of 4′-N,N-dimethylamino-substituted
analogues 2-(4′-N,N-dimethylaminophenyl)imidazopydridines
form an inclusion complex with β-CD.11,61 The mode of
encapsulation of 4′-N,N-dimethylamino-substituted analogues
is different from that of HPIP-b and HPIP-c. The 4′-N,N-
dimethylaminophenyl is encapsulated, and the benzimidazole/
imidazopyridine ring is present outside of the cavity.11,61,79 In
4′-N,N-dimethylamino analogues, also the monocations are
formed by protonation of heterocylic ring nitrogen. Because the
cationic part of the molecule is present outside of the cavity, the
cation binds with the β-CD cavity, and the solvation shell
around the cationic heterocyclic ring is intact. On the other
hand, in HPIP-b or HPIP-c, the heterocyclic ring enters the
hydrophobic cavity; this will remove the shell solvation around
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +91-3612582315. Fax +91-3612582349. E-mail:
gkrishna@iitg.ernet.in.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the Department of Science and
Technology (DST), India and the Council of Scientific and
Industrial Research (CSIR), India for financial support. Central
Instrumental Facility (CIF), IIT Guwahati is acknowledged for
the TCSPC and the proton NMR instruments and Gauhati
University for the 13C NMR spectral measurements. The
authors thank Dr. M. Hormi of Gauhati University for helping
obtain the 13C NMR. F.A.S.C. and S.K.B. thank CSIR for
research fellowships.
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DEDICATION
#Dedicated to Prof. Jack Saltiel on his 75th birthday.
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