Benzimidazole acrylonitriles as multifunctional push-pull chromophores: Spectral characterisation, protonation equilibria and nanoaggregation in aqueous solutions

Article abstract of DOI:10.1016/j.saa.2017.02.011

Heterocyclic donor-π-acceptor molecular systems based on an N,N-dimethylamino phenylacrylonitrile benzimidazole skeleton have been characterised and are proposed for potential use in sensing applications. The benzimidazole moiety introduces a broad spectrum of useful multifunctional properties to the system including electron accepting ability, pH sensitivity and compatibility with biomolecules. The photophysical characterisation of the prototropic forms of these chromophores has been carried out in both solution and on immobilisation in polymer films. The experimental results are further supported by computational determination of pK_a values. It is noticed that compound 3 forms nanoaggregates in aqueous solutions with aggregation-induced emission (AIE) at 600?nm. All the systems demonstrate spectral pH sensitivity in acidic media which shifts towards near-neutral values upon immobilisation in polymer films or upon aggregation in an aqueous environment (compound 3). The structure-property relationships of these functional chromophores, involving their spectral characteristics, acid-base equilibria, pK_a values and aggregation effects have been determined. Potential applications of the molecules as pH and biomolecular sensors are proposed based on their pH sensitivity and AIE properties.

Full text of DOI:10.1016/j.saa.2017.02.011

Accepted Manuscript
Benzimidazole acrylonitriles as multifunctional push-pull
chromophores: Spectral characterisation, protonation equilibria
and nanoaggregation in aqueous solutions
Ema Horak, Robert Vianello, Marijana Hranjec, Svjetlana
Krištafor, Grace Karminski Zamola, Ivana Murković Steinberg
PII:
DOI:
Reference:
S1386-1425(17)30099-9
doi: 10.1016/j.saa.2017.02.011
SAA 14933
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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28 October 2016
31 January 2017
4 February 2017
Please cite this article as: Ema Horak, Robert Vianello, Marijana Hranjec, Svjetlana
Krištafor, Grace Karminski Zamola, Ivana Murković Steinberg , Benzimidazole
acrylonitriles as multifunctional push-pull chromophores: Spectral characterisation,
protonation equilibria and nanoaggregation in aqueous solutions. The address for the
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ACCEPTED MANUSCRIPT
Benzimidazole acrylonitriles as multifunctional push-pull
chromophores: spectral characterisation, protonation
equilibria and nanoaggregation in aqueous solutions
Ema Horaka, Robert Vianellob, Marijana Hranjecc, Svjetlana Krištafora, Grace Karminski
Zamolac and Ivana Murković Steinberga*
aDepartment of General and Inorganic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 19, HR 10000 Zagreb, Croatia
bComputational Organic Chemistry and Biochemistry Group, Ruđer Bošković Institute, Bijenička
cesta 54, HR 10000 Zagreb, Croatia
c Department of Organic Chemistry, Faculty of Chemical Engineering and Technology
University of Zagreb, Marulićev trg 20, HR 10000 Zagreb, Croatia
*Corresponding author:
Ivana Murković Steinberg, Department of General and Inorganic Chemistry, Faculty of Chemical
Engineering and Technology, University of Zagreb, Marulićev trg 19, HR-10000 Zagreb, Croatia,
Phone No. ++38514597287; e-mail: ivana.murkovic@fkit.hr
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Abstract
Heterocyclic donor-π-acceptor molecular systems based on an N,N-dimethylamino
phenylacrylonitrile benzimidazole skeleton have been characterised and are proposed for
potential use in sensing applications. The benzimidazole moiety introduces a broad spectrum
of useful multifunctional properties to the system including electron accepting ability, pH
sensitivity and compatibility with biomolecules. The photophysical characterization of the
prototropic forms of these chromophores has been carried out in both solution and on
immobilisation in polymer films. The experimental results are further supported by
computational determination of pKa values. It is noticed that compound 3 forms
nanoaggregates in aqueous solutions with aggregation-induced emission (AIE) at 600 nm. All
the systems demonstrate spectral pH sensitivity in acidic media which shifts towards near-
neutral values upon immobilisation in polymer films or upon aggregation in an aqueous
environment (compound 3). The structure-property relationships of these functional
chromophores, involving their spectral characteristics, acid-base equilibria, pKa values and
aggregation effects have been determined. Potential applications of the molecules as pH and
biomolecular sensors are proposed based on their pH sensitivity and AIE properties.
Keywords: push-pull chromophores; benzimidazole; aggregation induced emission (AIE); pH
sensitivity; prototropic equlibrium
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1. Introduction
Intramolecular charge-transfer (ICT) organic systems based on electron donor-π-bridge-
electron acceptor structures (D-π-A) are widely used as chromophores and fluorophores in
advanced functional materials [1] and (bio)analytical or imaging applications [2].
The photophysical and chemical properties of such push-pull molecular systems are defined
by the position, number and electron donating and accepting strengths of donor and acceptor
groups and their chemical nature. Insight into the structure-property relationships allows fine
tuning of their functional properties [3-5]. In this respect D-π-A systems based on
heteroaromatic scaffolds such as styryl heterocycles [6-8] or imidazole derivatives [9] are
especially interesting due to the additional functional molecular features they bring to
chromophores: specific chemical and biological activity.
The benzimidazole moiety (BI) can serve as a multifunctional unit in such heteroaromatic
molecular systems: its electron accepting and π bridging properties combined with
chromogenic pH sensitivity/switching, metal-ion chelating properties and compatibility with
biomolecules makes the BI moiety an especially attractive building block in D-π-A systems
for different applications: optoelectronics and non-linear optics (NLO), photovoltaics, sensing
and bioimaging. Representative examples of BI based ICT chromo- and fluorophores include
materials for OLEDs [10], dye-sensitised solar cells (DSSCs) [11, 12] and molecular
chemosensors [13], sensors for pH [14-17] metal-ions [18, 19], anions [20], biomolecules
[21], and pH probes for bioimaging [22].
The structure-property impact of BI units within D-π-A heteroaromatic chromophores,
especially pH sensitivity, has been studied in detail spectroscopically and theoretically [23-
26] in potential sensing applications and for NLO pH switching. Studies involve
computational prediction and determination of pKa values and photophysical or non-linear
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properties and usually represent the first step in the design of novel multi-responsive
molecular systems.
As a result of their dipolar structure, the ICT compounds can also form self-assembled
functional molecular systems [27]. The cyano group, as a typical strong electron acceptor in
D-π-A systems strongly affects the spectral characteristics of the molecules (λabs and ε), as
well as the basicity of neighbouring nitrogen atoms and their corresponding pKa values [28].
Additionally, the presence of a cyano group in these chromophores has been identified as a
key factor in the creation of supramolecular interactions [29-32], leading to the formation of
fluorescent organic nanoparticles (FONs) that exhibit aggregation induced emission (AIE)
[33]. Aggregation induced emission is a photophysical phenomenon with great application
promise, related to certain organic molecules which exhibit stronger fluorescence in their
aggregated states than in the dissolved state.
Here we present multifunctional D-π-A molecular systems containing an N,N-dimethylamino
group as a pH sensitive donor group connected via a π linker (styryl) to the electron accepting
substituents: cyano group and the pH sensitive benzimidazole moiety (Fig. 1).
Fig. 1.
The photophysical characterisation and computationally supported determination of species
involved in prototropic equilibria, including their respective pKa values, have been performed
in order to better understand the effects of the D-π-A molecular structure on the UV-Vis
spectral properties, nanoaggregation and pH sensing potential of these chromophores.
The results are critically evaluated in the light of structurally similar multifunctional
compounds including cyanostilbenes [34], styrylbenzoxazoles [35] and cyanovinyl substituted
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benzimidazoles [11, 19] as promising candidates for functional application as AIE-based pH
probes, dye sensitizers in solar cells and as metal-ion sensors.
2. Materials and Methods
2.1. Reagents and instrumentation
Chemicals for synthesis and pure organic solvents were purchased from commercial suppliers
Acros, Aldrich or Fluka. Hydrochloride acid and potassium hydroxide were obtained from
Kemika d.d., Zagreb. Poly(vinylchloride) (PVC, high molecular weight) for thin film
preparation was purchased from Fluka, whilst dioctyl sebacate (DOS) and potassium
tetrakis(4-chlorophenyl)borate (PTCB) were obtained from Sigma-Aldrich. Polyester sheets
(Laser + Copier Film 210 x 297 mm, d = 0.1 mm) were purchased from Zweckform. Milli-Q
water was used for the preparation of aqueous solutions. pH was measured on commercially
available pH electrode Blue Line 17 pH, Schott AG, Mainz, Germany. Absorption spectra
were recorded by Cary 100 Scan Varian spectrophotometer. Fluorescence measurements were
carried out by Varian Cary Eclipse fluorescence spectrophotometer. Quartz cells of 1 cm path
length were used throughout measurements and absorbance and fluorescence values were
recorded at 1 nm. Wavelength scan was performed between 250 nm and 800 nm. Baseline
was recorded prior to each set of experiments in aqueous and non-aqueous solutions.
Emission spectra corrected for the effects of time- and wavelength-dependent light-source
fluctuations using a standard of Rhodamine 101, a diffuser provided with the fluorimeter and
the software supplied with the instrument. Dynamic light scattering (DLS) experiments were
conducted using Malvern Zetasizer Nano range light scattering instrument.
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2.2. Synthesis of benzimidazole derivatives
All chemicals and solvents were purchased from commercial suppliers Aldrich and Acros.
Melting points were recorded on SMP11 Bibby and Büchi 535 apparatus. NMR spectra were
1
13
measured in DMSO-d6 solutions using TMS as an internal standard. The H and C NMR
spectra were recorded on a Varian Gemini 300 or Varian Gemini 600 at 300, 600 and 150 and
75 MHz, respectively. Chemical shifts are reported in ppm (δ) relative to TMS. All
compounds were routinely checked by TLC with Merck silica gel 60F-254 glass plates.
The 2-benzimidazolyl substituted chromophores 1-4 (Fig. 2) were prepared in one-step
condensation of 2-cyanomethylbenzimidazole or 2-cyanomethyl-N-phenylbenzimidazole with
corresponding heteroaromatic aldehydes in absolute ethanol by adding a few drops of
piperidine [19, 36]. Detailed synthesis and structure properties of 1-4 can be found in
supplementary material.
Fig. 2.
2.3. Photophysical characterisation and acid-base properties
Basic photophysical properties of examined compounds were characterised by UV-visible
absorption and fluorescence spectroscopy. Spectra were recorded in solvents with different
ET(30) solvent polarity parameters [37]. The concentration of the chromophore solutions was
1x10−5 mol dm−3. All the solvents were of high purity grade. Excitation wavelengths were
determined from absorbance maxima. Quantum yields were determined by IUPAC method
using quinine sulphate as standard (Φ = 0.546). The characterisation of benzimidazole
derivatives 1-4 was conducted by diluting the stock solutions in methanol or ethanol
(methanol or ethanol volume fraction in diluted solutions did not exceed 1.5 %).
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Concentrations of stock solutions were as it follows: c(1)=2.18×10−3 mol dm−3;
c(2)=1.35×10−3 mol dm−3 c(3)=6.4×10−4 mol dm−3; c(4)=9.2×10−4 mol dm−3.
The pH titration experiments were carried out using 1x10−5 mol dm−3 solutions of the
compounds 1-4 in 0.1 M hydrochloric acid or 0.1 M potassium hydroxide solutions. The
adjustment of pH was achieved through the acid or base addition until the change of pH
within 0.5 units occurred. Following each addition of titrant, UV-visible absorption spectrum
was recorded after the time required keeping the drift of pH electrode within 0.1 pH units per
minute. Titrations were conducted at constant ionic strength I = 0.1 M, in potassium chloride
solutions. The pKa values were calculated as the x-coordinate of the inflection point of the
Boltzmann function (1) obtained from spectrophotometric titrations data:
퐴 −퐴
1
2
푦 =
(푥−푥 )/푑푥 + 퐴2
(1)
0
1+푒
Dynamic light scattering (DLS) experiments were carried out in aqueous (fw = 99%
water/ethanol) and in ethanol solution, c = 10 μM.
2.4. Theoretical calculations of pKa values
All of the molecular geometries were initially optimised in the gas-phase by the efficient and
accurate M06–2X/6–31+G(d,p) model. The thermal corrections were extracted from the
corresponding frequency calculations without the application of scaling factors. The final
single-point energies were attained with a highly flexible 6–311++G(2df,2pd) basis set using
the MP2 approach giving the MP2/6–311++G(2df,2pd)//M06–2X/6–31+G(d,p) model
employed here for the gas-phase free energies. To account for the solvation effects, we
reoptimized geometries employing the SMD polarisable continuum model [38] at the
(SMD)/M06–2X/6–31+G(d,p) level with all parameters corresponding to pure water, and
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calculated solvation free energies as a difference between the corresponding SMD and gas-
phase calculations at the same level of theory. The choice of such a computational setup was
prompted by our success in estimating both pKa and reaction thermodynamic values in
solution [39, 40]. pKa values were calculated in a relative fashion using AH + BREF → A– +
BREFH equation, and employing the following reference bases (BREFH): benzimidazole (pKa =
12.75 and 5.41) [41] for the deprotonation and protonation of this fragment, respectively, and
N,N-dimethylaniline (pKa = 5.15) [41] for the protonation of the dimethylamino group in
investigated systems. All of the calculations were performed using the Gaussian 09 software
[42].
2.5. Immobilisation of 3-4 in polymer films
Chromophores 3 and 4 were immobilised in plasticised PVC matrix starting from liquid
mixture prepared by modified published method [43]. The “cocktail' contained 67 mg PVC,
134 mg DOS (the plasticiser), 2.4 mg (1 eq) of PTCB and 1 eq of compound 3 or 4 in 1.5 mL
THF. Mixture was placed in ultrasonic bath for 15 min. Thin polymer films were prepared by
spreading 100 µL mixture onto a 2.5 x 2.5 cm solid transparent polyester sheet by spin-
coating technique. Obtained thin films were dried at room temperature for 18 h in dark. pH
response of immobilised chromophores was examined in buffer solutions, pH 2 to 12.
2.6. DNA interaction experiments
A polynucleotide calf thymus DNA was purchased from Sigma and Aldrich, and used without
further purification. Polynucleotide was dissolved in the BPE buffer (6mM Na2HPO4, 2mM
NaH2PO4, 1mM EDTA), pH=7.0. Stock solution of compound 3 was prepared in ethanol.
Respective aliquot of stock solution of 3 was added to the aqueous buffer (c = 1x10-5 mol dm-
3) and titration with DNA was conducted.
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Spectral measurements were performed in aqueous buffer solution (BPE buffer, pH=7.0, and
phosphate buffer, pH = 7.4). Absorbance and emission spectra were recorded after every
addition of DNA aliquot. The partial volume of ethanol in water did not exceed 0.1%.
3. Results and Discussion
3.1. Design and D-π-A structure of chromophores
Compounds 1 and 2 as parent molecules were converted into D-π-A molecular systems
(compounds 3 and 4) by introduction of strong electron donating N,N-dimethylamino group,
(D) into their phenylacrylonitrile benzimidazole conjugated skeleton.
Fig. 3.
In terms of D-π-A arrangement the structures of chromophores 3 and 4 are shown in Fig. 3. In
compound 3 the −NMe2 as a strong donor group (D) is connected via π linker (styryl) to the
very strong acceptor group A1 (cyano) and a weaker acceptor A2 (benzimidazole unit)
forming a D-π-A1-A2 structure. In compound 4, the benzimidazole moiety is modified with
bulky electron donating phenyl substituent (D1) which increases the electron density of A2.
All structures contain acid-base active sites as a part of either donor or acceptor moiety and
are marked in Fig. 3. These are amino nitrogen atom (in donor group D) and imino (azole)
nitrogen atom (in acceptor group A2) as protonable sites in 3 and 4. The NH group on the
benzimidazole ring in 3 is marked as deprotonable site.
Structural analogues of 3 previously reported in the literature included N,N-dimethylamino
styryl benzimidazole [25] (D-π-A2), N,N-dimethylamino pyridyl acrylonitrile [32] (D-π-A1-
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pyrydyl), cyanostilbene derivatives [34] (D-π-A1-(phenyl or phenoyl)) and N,N-diethylamino
analogue of 3 with carboxyl group attached to BI moiety [11].
The proposed donor- π- acceptor structures were used as a basis for discussion of structure-
property relationships of 1-4 determined in this study (photophysical properties of prototropic
species and the corresponding pKa values).
3.2. Photophysical properties of chromophores 1-4
The chromophores were studied by UV-Vis absorption and fluorescence spectroscopy in
different solvents. Absorption spectra of compounds 1-4 in ethanol are shown in Fig. 4a, and
their basic photophysical properties in ethanol are summarised in Table 1.
Fig. 4.
Table 1.
Compounds 1 and 2 show the main absorption bands with maxima at 350 nm and 335 nm
respectively. As expected, introduction of an N,N-dimethylamino group (−NMe2) into 1 and 2
caused a bathochromic shift of 75 nm and 65 nm and considerable increase of molar
absorption coefficients, as observed in compounds 3 and 4, respectively (Table 1).
Both effects can be explained by the appearance of a strong intramolecular charge transfer
(ICT) due to D-A interactions involving the electron donating −NMe2 group.
As a result of its D-π-A1-A2 structure with enhanced push-pull effects, compound 3 shows
the greatest red-shift with an absorption maximum at 425 nm, and a very high molar
absorption coefficient of 43 910 dm3 cm–1 mol–1. In 4, ICT effects are less pronounced due to
D-π-A1-A2-D1 structure in which the electron donating phenyl substituent (D1) reduces the
electron accepting power of the acceptor moiety. Previously reported N,N-dimethylamino
styryl benzimidazole [25] has an absorption maximum at 374 nm. Introduction of a strong
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electron-withdrawing cyano group into its skeleton (compound 3) results in a 50 nm
bathochromic shift. All compounds show modest fluorescence in ethanol with quantum yields
less than 0.01 and emission maxima that range from 444 nm to 491 nm. Due to relatively low
fluorescence intensity in ethanol the compounds are not readily promising candidates for
fluorimetric analytical applications.
The absorption spectra of 1-4 in aqueous solutions are shown in Fig. 4b, and the summary of
absorption spectroscopy data in solvents of different polarity are presented in Table 2.
The absorption maxima of compounds 1 and 2 (λabs) recorded in solvents of varying polarity
indicate a weak negative solvatochromic effect. A change of solvent from toluene to water
induced a blue-shift in the absorption maxima of Δλ(1) = 18 nm and Δλ(2) = 10 nm.
Compound 3 in water showed unusual spectral characteristics (Fig. 4b) indicating the
presence of specific solvent effects, whilst 4 exhibited a very small positive solvatochromism
with absorption maxima ranging from 390 nm in nonpolar aprotic toluene to 400 nm and 404
nm in hydrogen bond donating solvents, ethanol/methanol and water respectively. The reason
for the different solvatochromic responses of 1-4 is most likely due to their different D-A
structures, position and nature of ionisable sites (Fig. 3), and the corresponding polarity of the
molecules in their respective ground and excited states. The small blue shift observed in 1 and
2 on increased solvent polarity suggests that these molecules possess a greater dipole moment
in their ground state than in their respective excited states, whereas the red shift of 4 can be
explained by a greater dipole moment in the excited state, leading to stronger stabilisation in
solvents of higher polarity. A similar trend is observed for molar absorption coefficients (ε)
which generally decrease on going from toluene to water for 3 and 4. The spectral bandwidth
expressed as FWHM (Full Width at Half Maximum) value is fairly constant for 1 and 2 in all
solvents (around 70 nm), but for 3 (except in water) and 4 increases with solvent polarity, as
shown in Table 2. In comparison with other compounds, 3 has the highest values of ε and λabs
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in all solvents (except in water), which can be attributed to its stronger ICT character as
discussed earlier based on its D-π-A1-A2 structure.
Table 2.
It is evident that compound 3 show significantly different spectral characteristics in water in
comparison to other solvents. Namely, 3 is yellow in all solvents except in water. Water
solution is visually colourless and transparent, and spectrally it shows a sharp non-
symmetrical (red-tailed) absorption band (FWHM value of 31 nm) with a maximum at around
350 nm (Fig. 4b). The water solution absorption peak wavelength λabs of 3 is blue-shifted by
approximately 70 nm in comparison to other solvents. The ICT character of 3 becomes less
pronounced in water solutions and is comparable to the spectral characteristics of compound
1, its non-ICT analogue. These findings strongly indicate the presence of dye nanoaggregates,
formed by the specific intermolecular interactions in aqueous environment. The shape of the
absorption spectrum of 3 in water indicates presence light scattering effects on nanoparticles
[44]. This was confirmed by Tyndall effect which occurred on visually transparent solution of
3 using a laser beam, as shown in Fig. S1A. In addition, the presence of nanometer and
micrometer sized aggregates of 3 in aqueous solutions (fw = 99 %, c = 10 µM) was confirmed
by dynamic light scattering experiments (Fig S1B).
It was also noticed that the water solution of 3 (colourless under ambient light) shows orange-
red emission under UV light. Hence, the effects of nanoaggregation on spectroscopic
properties of 3 were further studied in mixed solvent solutions (water-ethanol).
3.3. Aggregation induced emission in aqueous solutions
The absorption and emission characteristics of 3 were investigated in water-ethanol mixtures
of varying volume fraction of water, fw. The resulting spectra are shown in Fig. 5a.
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Fig. 5.
Compound 3 shows orange-red fluorescence emission at 600 nm in water, which gradually
disappears upon addition of ethanol (Fig. 5). At a water fraction of fw = 0.80 virtually no
florescence emission at 600 nm is observable. The intensity of orange emission in aqueous
solution (fw = 99 %) is approximately 35-fold the emission in ethanol at 600 nm. This
photophysical phenomenon can be explained by aggregation induced emission, as first
reported by Tang et al. [45] and Park et al. [30]. Unlike most organic fluorophores, some
organic molecules exhibit strong fluorescence upon aggregation in poor solvents or in the
solid-state. Different mechanisms have been proposed to explain AIE including restriction of
intramolecular rotation (RIR), J-aggregate formation, twisted intramolecular charge transfer
(TICT) and others [33].
Since a previously reported analogue of 3, N,N-dimethylamino styryl benzimidazole [25],
does not show aggregation in water, it is clear that the presence of the cyano group in 3 is
responsible for intermolecular changes leading to the observed AIE. It is known that
supramolecular interactions, especially hydrogen bonds and other dipole-dipole interactions
involving cyano groups [46] may be responsible for the suppression of intramolecular
motions in crystals resulting in tight molecular arrangements of increased rigidity leading to
AIE phenomena [32, 47]. However, compound 4 also contains a cyano group but does not
show aggregation in water. The BI moiety in 4, with its bulky electron donating phenyl
substituent (D1), disturbs the molecular planarity and prevents certain dipole-dipole
interactions that are possibly responsible for the aggregation observed in compound 3. It is
interesting to note that the observed blue-shift in the absorption band of 3 on going from
ethanol (440 nm) to aqueous solution (350 nm) can easily be associated with the formation of
non-emissive H-aggregates resulting from π–stacking interactions. However, in contrast to
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that, 3 shows red-shifted and significantly increased emission in nanoaggregated form. This
can be related to a less common conformation of emissive H-aggregates [48] where the
presence of the bulky and polar cyano group is known to affect the parallel face-to-face
intermolecular interactions [30].
3.3. Protonation equilibria and pKa values
3.3.1. Protonated species of 1-4 in ethanol
In order to define the species involved in neutral–monocation equilibria, spectral
characterisation was first performed in acidified ethanol solutions to avoid possible
complications due to observed nanoaggregation in aqueous solutions.
Absorption and emission spectra of neutral (M) and monoprotonated species (M+) of 1-4 are
shown in Fig. 6 and the summary of spectral properties of neutral and protonated
chromophores in ethanol are presented in Table S1. Excitation and emission spectra of 3 and
4, in neutral and protonated forms in ethanol, are shown in Fig. S2.
Fig. 6.
The absorption spectra of protonated species of compounds 1 and 2 in ethanol (Fig. 6) show
no significant change of the longest wavelength peak, whilst compounds 3 and 4 exhibit
bathochromic shifts with increased extinction coefficients. Since monoprotonated species of 3
and 4 contain two protonable sites (N,N-dimethylamino nitogen and imino nitrogen on BI
moiety) the question is which nitrogen atom is more basic and would be protonated first.
It is generally known that protonation of a donor group (amino) in a push-pull system is
reducing its electron-donating character and decreasing conjugation which results in a blue
shift of the absorption spectrum and decreased extinction coefficient. On the other hand,
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protonation of acceptor moiety in a push-pull system is enhancing acceptor’s electron-
withdrawing character and results in a red-shift and increased extinction coefficients of
molecules. The spectroscopic results indicate that protonation in 3 and 4 occurs first on imino
nitrogen atom on BI moiety (the acceptor group), which is in agreement with previously
reported styryl analogue of 3 [25] and the hydrochloride monohydrate of 3 whose crystal
structure also confirmed that protonation occurs on imino nitrogen [36]. This is further
corroborated by the computational results presented later.
Protonation of BI moiety on 1 and 2 (which are not typical ICT molecules) is not related to
significant changes in absorption spectra. The lone electron pair on imino nitrogen occupies
sp2 hybrid orbital and is not involved in the π electron system. Its protonation does not
considerably affect HOMO-LUMO energy levels, so the absorption spectra remain virtually
unchanged. Fluorescence emission of neutral species in ethanol is relatively weak (Table 1).
Protonation of 1 and 2 leads to increased fluorescence emission without shift of emission
wavelength.
3.3.2. Spectroscopic pH titrations of 1-4 in aqueous solutions
General protonation-deprotonation equilibria and corresponding pKa values of compounds 1-4
are shown in Scheme 1. (Note: under investigated conditions compounds 1 and 2 do not form
M2+ species, while compounds 2 and 4 do not form M− species)
Scheme 1.
The spectroscopic pH titrations were performed in aqueous solutions in the pH range 1.5 to
13.0 and the resulting absorption spectra for 3 and 4 are shown in Fig. 7a and 7b, respectively.
The spectral identification of all species involved in respective protonation-deprotonation
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equilibria of 1-4 in aqueous solutions was performed, and the summary of spectroscopic
properties is presented in Table S2. Spectral properties of dicationic and anionic
(deprotonated) species are estimated from spectra obtained at pH = 1.0 and pH = 14.0,
respectively.
As shown in Fig. 7a titration spectra of 3 show three distinct pH ranges corresponding to: i)
protonation of neutral form (pH range 1.5 to 5); ii) neutral form (aggregate) at pH values
ranging from approximately 5 to 8, and iii) deprotonation of neutral form (pH range 9 to 13).
The protonation of monoprotonated form to dication form (amino-protonated species) occurs
at pH values below 2 and is accompanied with blue shifted absorption maxima at 335 nm. The
pH titration spectra of 4 show no evidence of aggregation and correspond to equivalent
spectra obtained in ethanolic solutions.
Monoprotonated forms of both, 3 and 4 have considerably higher molar extinction
coefficients than the corresponding neutral species, as expected from their ICT structure since
protonation occurs at the acceptor moiety.
Fig. 7.
3.3.3. Experimental and theoretical pKa values in aqueous solutions
The pKa values are experimentally determined or estimated from spectroscopic titration data.
Fig. 8 shows examples of plots used for experimental determination of pKa values.
Fig. 8.
In order to rationalize the acid/base features of studied systems in aqueous solution we
performed a computational analysis employing the implicit SMD solvation. The calculated
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pKa values (Table 3) show remarkable agreement with experimental data, which lends
credence to the applied computational model. In comparison with the six precisely measured
values, the average absolute deviation is only 0.6 pKa units, which is remarkable for this kind
of calculations. Nevertheless, we must emphasize that it would be even much lower if it
would not include a 2 pKa difference in the pKa2(3) value that we attribute to the reported
aggregation of this system in water, which complicates experimental pKa determination.
Table 3.
Interestingly, the first protonation of all four systems 1–4 occurs on the benzimidazole imino
nitrogen. This might be expected considering that benzimidazole (pKa = 5.41) is slightly more
basic than N,N-dimethylaniline (pKa = 5.07) [41], together with the fact that the cyano group
in 3 and 4 is in a direct resonance interaction with the latter fragment, thus reducing its
basicity. In the parent system 1, imino nitrogen protonation is associated with pKa2 = 3.0
making it significantly less basic than benzimidazole. This is a general trend in all 1–4, which
strongly indicates that under normal conditions all four investigated systems are
predominantly present as unionized neutral molecules, which is a significant observation. We
attribute this trend to the presence of the electron withdrawing styryl moiety, which
diminishes the basicity of the benzimidazole fragment. This effect is slightly compensated by
adding the electron donating –NMe2 group in 3 and 4, where the corresponding pKa2 values
increase to 4.2 and 3.7, but still not enough to make these systems more basic than
benzimidazole. As expected, if we remove the strongly acidifying cyano group [49, 50] from
1 (model system I, Table 3) the corresponding pKa value increases to 5.8.
The second protonation of 3 and 4 occurs at the dimethylamino group and, interestingly, it
requires only slightly stronger acids than those able to allow the corresponding monocations.
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pKa1 values for 3 and 4 assume 1.1 and 2.0 (Table 3), which could be achieved with two
equivalents of already moderately strong acids. As mentioned, dimethylamino positions are in
a direct resonance with the attached cyano group, so these positions in 3 and 4 are
significantly less basic than N,N-dimethylaniline (pKa = 5.07). Differing effect of the –CN
group is evident in the changes of bond distances induced upon protonation. For example,
after the imino protonation of 3 to 3+, the C–N distance of the cyano group is not changed and
remains the same at 1.162 Å, but is subsequently reduced to 1.159 Å after amino protonation
of 3+ to 32+. In line with previous discussion, dication I2+ is even more easily attainable (pKa1
= 3.3). On the other hand, the unsubstituted benzimidazole amino groups in 1 and 3 are prone
to deprotonation. Relative to benzimidazole deprotonation (pKa = 12.75) [41], the presence of
the cyano group increases the acidity of 1 and 3 by two orders of magnitude to the pKa3 values
of 10.4 and 10.3 (Table 3), which is notable, yet not enough to make these systems
monoanions under normal conditions. Since I is deprived of the cyano group, this effect is
absent (pKa3 = 12.5).
In concluding this section, it is worth emphasizing that the calculations underline a significant
electronic effect of the cyano group in affecting acid/base properties of 1-4, and convincingly
demonstrate that all investigated systems are neutral molecules under normal conditions,
which become monoprotonated exclusively on the benzimidazole imino with moderately
strong acids (pKa values between 2 – 4).
The neutral species of 3 forms nanoaggregates in aqueous solution. The deaggregation
process can be triggered either by protonation or deprotonation of molecules occurring at pH
values lower than 5 or higher than 8, respectively. It is important to note that the aggregation-
deaggregation process is reversibly switched by changing the pH of the aqueous solution,
opening up interesting potential applications for compound 3 in AIE-based pH sensing and
imaging. Also, it has previously been shown that the hydrochloride monohydrate of 3
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interacts with DNA molecules [36], a result which should now be explored further in light of
the spectrally responsive and pH sensitive deaggregation processes we report here.
3.4. pH sensitivity of chromophores in polymer matrix
Thin polymer films based on plasticised PVC containing immobilised 3 and 4 were fabricated
and initially tested as optically pH responsive materials for ion-selective optodes. The spectral
and visual responses to pH, as well as the corresponding calibration plot of immobilised 3 are
shown in Fig. 9.
Fig. 9.
Table 4.
Absorption and emission properties and the corresponding apparent pKa values of neutral and
monocation forms of immobilised 3 and 4 are summarised in Table S3. The spectral response
of the immobilised chromophores corresponds to ethanolic solutions and no aggregation of 3
was noticed. The apparent pKa values increased by nearly 3 pKa units in comparison to their
aqueous values. Such changes are expected due to a number of known parameters affecting
the complex heterogeneous ion-exchange equilibria in ion-selective optodes [51].
3.5. Effect of DNA on aggregation
The effect of DNA on the aggregation/deaggregation equilibrium of 3 has been studied. The
absorption and emission spectral responses obtained in a ct-DNA titration experiment are
shown in Fig. 10. In the presence of DNA the absorbance and emission intensities of the
nanoaggregated form of 3 (at 350 nm and 600 nm, respectively) decrease. The corresponding
titration plot is shown (Fig. 10, inset). The interaction between 3 and the ct-DNA molecules
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results in quenching of AIE. The interaction is most likely based on structural compatibility of
the planar BI moiety and its ability to incorporate between adjacent base pairs of the DNA
molecule during the intercalation process [36, 52].
Fig. 10.
4. Conclusion
The benzimidazole-based push-pull molecular systems presented in this work exhibit pH
induced spectral responses in the visible region with relatively high molar absorption
coefficients. In general, the fluorescence properties of the molecules are not promising for
applications requiring high quantum yields, with the exception of 3 and the corresponding
aggregation-induced emission. All the chromophores demonstrate pH sensitivity with pKa
values in acidic (2.1-3.0) and basic (around 10) ranges, corresponding to neutral-monocation
and neutral-anion equilibria. Computational analysis aided in rationalising acid/base features
of the systems investigated, and demonstrated that all compounds are unionised under normal
conditions, but become monoprotonated exclusively on the benzimidazole imino nitrogen
with only moderately strong acids (pKa values between 2-4). However, the apparent pKa
values of 3 and 4 when immobilised in polymer films shifted to 6.3, enabling possible future
application of these chromophores as physiologically compatible pH sensitive materials. An
important finding is the ability of 3 to form emissive nanoaggregates in solution triggered by
either solvent composition or the pH value of the aqueous solution. Its near-neutral pH-
switchable aggregation/deaggregation is accompanied by absorption and emission changes
making it promising for pH sensing and imaging applications. An additional advantage for
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biological applications is the excitation at 350 nm resulting in a long wavelength emission at
600 nm.
Further studies exploring the effects of interactions of the BI moiety with biomolecules and
metal-ions on AIE are underway in our laboratory.
Acknowledgments
This work was supported by the Croatian Science Foundation under grant number IP-2014-
09-3386 entitled Design and synthesis of novel nitrogen-containing heterocyclic fluorophores
and fluorescent nanomaterials for pH and metal-ion sensing which is gratefully
acknowledged. We would like to thank Dr. Maja Dutour Sikirić and Dr. Darija Domazet
Jurašin of Ruđer Bošković Institute, Zagreb, for use of the DLS instrumentation and for
characterisation of the nanoaggregates. We would also like to thank Dr. Matthew D. Steinberg
for reviewing the manuscript.
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Figure captions
Fig. 1. D-π–A molecular system based on benzimidazole derivative.
Fig. 2. Synthesised benzimidazole derivatives 1-4.
Fig. 3. D-π-A arrangement and protonable – deprotonable sites of (a) 3 and (b) 4.
Fig. 4. Absorption spectra of compounds 1-4, c = 1 x 10-5 M, in (a) ethanol and (b) water.
Fig. 5. (a) Absorbance and emission spectra of compound 3 in water and in ethanol, λexc = 350
nm; (b) Absorbance of 3 in ethanol/water mixtures recorded at 425 nm; (c) Photographs of 3
in water/ethanol mixtures under ambient light and under UV lamp (365 nm), volume fraction
of ethanol in mixed solutions, fEtOH = 0 - 40%, c (3) = 1 x 10-5 M.
Fig. 6. Absorption and emission spectra of compounds 1 - 4, c = 1 x 10-5 M, in neutral and
protonated form in ethanol. (Excitation wavelength at absorption maximum of corresponding
species.)
Scheme 1. General scheme of acid-base equilibria 1-4.
Fig. 7. Spectroscopic pH titration of (a) 3 and (b) 4, c = 1 x 10-5 mol dm-3 in aqueous
solutions and corresponding equilibria.
Fig. 8. Absorbance data plotted versus pH for (a) 3 and (b) 4 at selected wavelengths.
Fig. 9. (a) Absorbance spectra of chromophore 3 immobilised in plasticised PVC film at
different pH values; (b) calibration plot of immobilised chromophore 3 (absorbance change in
response to pH at 495 nm).
Fig. 10. a) Absorption and b) emission spectra (λexc = 350 nm) of 3 in aqueous solution
(buffer pH = 7.0) upon titration with DNA.
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Fig. 1
Fig. 2
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Fig. 3
Fig. 4
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Fig. 5
29