Complexes of carboxylato pillar[6]arene with Brooker-type merocyanines: Spectral properties, pKa shifts and the design of a displacement assay for trimethyl lysine

Article abstract of DOI:10.1016/j.saa.2021.119455

The supramolecular complexes of three strongly solvatochromic dyes, Brooker's merocyanine (M1) and its two derivatives (M2, M3) with carboxylato pillar[6]arene (WP6) were studied in aqueous solutions. The dye-WP6 mixtures were described in terms of four equilibrium reactions: the acidic dissociations of the pyridinium phenols into the zwitterionic phenolates, the acidic dissociations of the complexed phenols, the bindings of the phenol form dyes to WP6 and the bindings of the phenolates to WP6. The equilibrium constants were determined by an analysis of the absorption spectra. It was found that the acidity of the phenol form merocyanines were largely reduced on complexation, pK_a shifts of 1.1–1.6 units were observed. In neutral solutions, the complexes of the phenol forms of M1 and M2 were dominant, in contrast to the more acidic M3 (a dibromo derivative), of which the phenolate complex was more stable. Comparing the spectral properties, the binding constants and the pK_a-s of the dye-WP6 complexes, the complex M3?WP6 was chosen to be tested as a displacement assay. It was demonstrated that this complex functioned as a colorimetric indicator displacement assay which discriminated trimethyl lysine from other lysine derivatives.

Full text of DOI:10.1016/j.saa.2021.119455

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 252 (2021) 119455
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Complexes of carboxylato pillar[6]arene with Brooker-type
merocyanines: Spectral properties, pKa shifts and the design of a
displacement assay for trimethyl lysine
Dóra Hessz a, Stella Bádogos a, Márton Bojtár b, István Bitter c, László Drahos d, Miklós Kubinyi a,
⇑
a Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, 1521 Budapest, Hungary
b ‘‘Lendület” Chemical Biology Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences, 1519 Budapest, Hungary
c Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, 1521 Budapest, Hungary
d Institute of Organic Chemistry, Research Centre for Natural Sciences, 1519 Budapest, P.O.B. 286, Hungary
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
The complexation of three pyridinium
phenolate betain dyes with the water
soluble pillararene WP6 was studied.
Both the phenol and the phenolate
forms of the dyes form stable
complexes with WP6.
The pKa values of the phenol forms of
the dyes increase by 1.1–1.6 units on
complexation.
One of the dye-WP6 complexes
functioned as an indicator
displacement assay for Me3Lys.
a r t i c l e i n f o
a b s t r a c t
Article history:
The supramolecular complexes of three strongly solvatochromic dyes, Brooker’s merocyanine (M1) and
its two derivatives (M2, M3) with carboxylato pillar[6]arene (WP6) were studied in aqueous solutions.
The dye-WP6 mixtures were described in terms of four equilibrium reactions: the acidic dissociations
of the pyridinium phenols into the zwitterionic phenolates, the acidic dissociations of the complexed
phenols, the bindings of the phenol form dyes to WP6 and the bindings of the phenolates to WP6. The
equilibrium constants were determined by an analysis of the absorption spectra. It was found that the
acidity of the phenol form merocyanines were largely reduced on complexation, pKa shifts of 1.1–1.6
units were observed. In neutral solutions, the complexes of the phenol forms of M1 and M2 were dom-
inant, in contrast to the more acidic M3 (a dibromo derivative), of which the phenolate complex was
more stable. Comparing the spectral properties, the binding constants and the pKa-s of the dye-WP6 com-
plexes, the complex M3ꢁWP6 was chosen to be tested as a displacement assay. It was demonstrated that
this complex functioned as a colorimetric indicator displacement assay which discriminated trimethyl
lysine from other lysine derivatives.
Received 6 October 2020
Received in revised form 5 January 2021
Accepted 5 January 2021
Available online 8 January 2021
Keywords:
Pyridinium phenolate dye
Water-soluble pillararene
Solvatochromism
Host–guest complex
Acidic dissociation constant
Hydrophobic effect
Indicator displacement assay
Ó 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Brooker’s merocyanine (M1 in Fig. 1) is a pyridinium phenolate
dye which exhibits an extremely strong solvatochromic behavior
⇑
Corresponding author.
E-mail address: kubinyi@mail.bme.hu (M. Kubinyi).
https://doi.org/10.1016/j.saa.2021.119455
1386-1425/Ó 2021 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Dóra Hessz, S. Bádogos, Márton Bojtár et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 252 (2021) 119455
Fig. 1. The phenolate and phenol forms of Brooker’s merocyanine. The phenolate is
a hybride of a zwitterionic and a neutral mesomeric structure.
Fig. 2. The structures of the merocyanine dye guests and the pillararene host.
stituents in the phenol ring, was expected to lower the pKa signif-
icantly [32], for which zwitterion M3 was hoped to form a stable
complex with WP6 under neutral conditions. As a part of this work,
the pKa values of the free merocyanines and their WP6 complexes
were determined in spectroscopic experiments and the results
confirmed this hypothesis. Therefore, the M3–WP6 complex was
chosen to be tested as a displacement assay. The performance of
the assay was tested on the discrimination of lysine and its methy-
lated derivatives.
[1]. The absorption band of M1 appears at 442 nm in water and it
shifts to 620 nm in CHCl3 [2]. This negative solvatochromism of the
dye (hypsochromic shift with increasing solvent polarity) shown in
the majority of solvents, reverses in solvents of low polarity [3]. In
the generally accepted interpretation of the solvatochromic prop-
erties of M1, the structure of the dye is described as a hybrid of a
zwitterionic benzenoid and a neutral quinonoid canonical struc-
ture. In weakly polar solvents the hybrid is weighted toward the
neutral form, in polar solvents the zwitterionic form is dominant.
Theoretical calculations provided insightful information on how
solute–solvent interactions affect the molecular geometry, the
charge distribution and the S0 ? S1 excitation energy of the M1
dye solute [4–6]. Exploiting the high sensitivity of M1 to the local
environment, it is frequently applied as a solvatochromic probe in
liquid structure studies [7–9].
2. Materials and methods
2.1. Synthesis
The pillararene host WP6 was synthesized using the method of
Huang et al. [33]. Brooker’s merocyanine M1 was prepared from 1-
methylpicolinium iodide and 4-hydroxybenzaldehyde by Knoeve-
nagel condensation, as described in Ref [34]. The two new mero-
cyanine dyes, M2 and M3, were also prepared by Knoevenagel
condensation reactions, from the corresponding N-substituted
picolinium salt and benzaldehyde. The starting materials used in
the syntheses were commercial products. The NMR spectra were
recorded on a Bruker Avance DRX-500 spectrometer. The exact
mass measurements were performed with a Waters Q-TOF Premier
mass spectrometer using electrospray ionization in positive mode.
With its aromatic rings and oppositely charged ends, zwitteri-
onic M1 can coordinate to macrocyclic receptors in multiple ways.
So far, the complexes of M1 with cyclodextrins (CD-s), cucurbit[8]
uril (CB8) and a calix[4]pyrrol (CP) have been described. a-, b-, c-
and methyl-b-CD form 1:1 inclusion complexes with M1 [10–12].
The complex of M1 with b-CD is more stable than its complexes
with
a-and c-CD. As a consequence of the higher stability, the
trans–cis photoisomerization of the protonated form of M1 is hin-
dered in the b-CD complex of the dye [12,13]. CB8 accommodates
two M1 molecules which are aligned head-to-tail in its cavity and
which dimerize in a photochemical reaction [14,15]. The M1-CP
complex is held together by hydrogen bonds between the pheno-
late oxygen atom of M1 and the pyrrol NH groups of CP [16]. In
acetonitrile, this complex works as a colorimetric indicator dis-
placement assay for the basic anions Fꢂ, Clꢂ and H2PO4ꢂ.
In the present work, the complexes of three merocyanines, M1-
M3 in Fig. 2 with the water soluble pillararene macrocycle WP6
were studied. M2 and M3 were new compounds. Our final goal
was to construct an indicator displacement assay (IDA) for the
detection of cationic biomolecules, exploiting the high affinity of
the multiple negatively charged carboxylato pillararenes to bind
positively charged organic species [17–30].
The protic equilibria between the cationic phenol and the zwit-
terionic phenolate forms of the merocyanines was an important
aspect of the design. The pKa of the phenol form of M1 is 8.57
[31]. Thus, the phenolate form of M1, the spectral properties of
which are more sensitive to complexation, is dominant only in
basic media - a disadvantage with respect to potential applications
in biological samples. The phenyl substituent in M2 was expected
to modify the binding constant with WP6 host, but not the acidity
of the dye. In contrast, the introduction of the two bromo sub-
2.1.1. (E)-4-(2-(1-phenylpyridin-1-ium-4-yl)vinyl)phenolate (M2)
To
a solution of 1-phenylpicolinium chloride (411 mg,
2.0 mmol) and 4-hydroxybenzaldehyde (305 mg, 2.5 mmol, 1.25
equiv) in 15 mL methanol was added a few drops of piperidine.
The reaction mixture was refluxed overnight then the solvent
was evaporated. The crude product was crystallized from a mixture
of 10 mL acetone and 10 mL diethyl ether, filtered and washed
thoroughly with acetone and ether. The formed chloride salt (phe-
nol form) was suspended in 5 mL water, then an equivalent
amount of 1 mol/L NaOH solution was added (1.56 mL based on
the chloride salt). The brown suspension became deep red and
was filtered, washed three times with cold water and dried to yield
423 mg (77%) purple crystalline solid. M.p. 230–232 °C.
1H NMR (500 MHz, DMSO d6): d 8.91 (d, J = 5.8 Hz, 2H), 8.07–
8.01 (m, 3H), 7.80 (d, J = 7.3 Hz, 2H), 7.69–7.65 (m, 3H), 7.58 (d,
J = 8.2 Hz, 2H), 7.18 (d, J = 15.9 Hz, 1H), 6.70 (d, J = 8.2 Hz, 2H).
13C NMR (126 MHz, DMSO d6, TFA was added to the solution to
increase solubility): 160.55, 154.63, 143.75, 142.94, 142.44, 130.85,
130.74, 130.25, 126.43, 124.43, 123.13, 119.60, 116.23.
HR-MS calculated mass for C19H16NO [M + H]+ m/z = 274.1232,
found 274.1236.
2

Dóra Hessz, S. Bádogos, Márton Bojtár et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 252 (2021) 119455
2.1.2. (E)-2,6-dibromo-4-(2-(1-phenylpyridin-1-ium-4-yl)vinyl)
phenolate (M3)
To
a solution of 1-phenylpicolinium chloride (250 mg,
1.21 mmol) and 3,5-dibromo-4-hydroxybenzaldehyde (508 mg,
1.82 mmol, 1.5 equiv) in 10 mL methanol was added a few drops
of piperidine. The reaction mixture was refluxed overnight then
the solvent was evaporated. The crude product was crystallized
from 20 mL acetone, filtered and washed thoroughly with acetone.
The formed chloride salt (phenol form) was suspended in 5 mL
water, then an equivalent amount of 1 M NaOH solution was added
(1.06 mL based on the chloride salt). The brown suspension
became deep red and was filtered, washed three times with cold
water and dried to yield 453 mg (87%) purple crystalline solid.
M.p. 256–258 °C.
1H NMR (500 MHz, DMSO d6): d 8.70 (d, J = 6.8 Hz, 2H), 7.88
(d, J = 15.3 Hz, 1H), 7.82–7.77 (m, 6H), 7.68 (t, J = 6.8 Hz, 2H),
7.63 (t, J = 7.0 Hz, 1H), 6.88 (d, J = 15.3 Hz, 1H).
Fig. 3. Scheme of synthesis of merocyanine dyes M1-M3.
3.2. Merocyanine dye – WP6 systems
13C NMR (126 MHz, DMSO d6, TFA was added to the solution to
increase solubility): d 153.71, 152.68, 144.22, 142.41, 139.15,
132.22, 130.95, 130.22, 129.86, 124.43, 123.59, 122.95, 112.31.
3.2.1. Reaction scheme
The properties of the merocyanine dye – pillararene systems
were described in terms of four equilibrium reactions (see Fig. 4):
HR-MS calculated mass for
C19H14NOBr2 [M +
H]+ m/
z = 429.9442, found 429.9446.
(i) the acidic dissociation of the cationic pyridinium phenol dye
producing the phenolate (i.e. the zwitterionic) species;
(ii) the binding of the phenol form dye guest to the to the pil-
lararene host;
(iii) the binding of the phenolate (zwitterionic) dye guest to the
to the pillararene host;
2.2. UV–VIS spectra of merocyanine dyes and their WP6 complexes
The samples for these experiments were solutions of the pure
dyes in Britton Robinson buffers and solutions of dye-WP6 mix-
tures in Britton Robinson buffers and in 0.2 mol/L aqueous NaOH.
The ionic strengths of the buffers were adjusted to 0.2 mol/L with
appropriate amounts of KCl [35]. The spectra were recorded on an
Agilent 8453 diode array UV–VIS absorption spectrometer. All the
experiments were carried out at 25 °C.
(iv) the acidic dissociation of the complex of the phenol form dye
with the pillararene host.
The 1:1 stoichiometry of the MꢂWP6 complexes was confirmed
by the Job plots of the absorbance data of the three dye-pillararene
systems (see Figs. S7-S9).
2.3. Calculation of equilibrium constants
We note that WP6 was considered fully dissociated in the dye-
WP6 mixtures since only samples of pH ꢃ 6.5 were used. In more
acidic (pH 6.0 in Ref. [19]) solutions, partially dissociated WP6
appears which precipitates [33].
As shown in Fig. 4, reactions (i)-(iv) constitute a cyclic scheme.
In principle, any of the four equilibrium constants in the cycle can
be obtained from the other three K values. The accuracy of an indi-
rectly determined equilibrium constant is, however, limited by the
error propagation. Therefore, we attempted to determine the four
equilibrium constants in separate experiments.
The equilibrium constants (the acidic dissociation constants of
the pure dyes and their WP6 complexes and the binding constants
of the dye-WP6 complexes) were determined by a least square fit-
ting to the absorption spectra of dye solutions measured at differ-
ent pH values or at different pillararene concentrations. The dye
formed four absorbing species, the free phenol M+, the free pheno-
late M (this corresponds to the zwitterion), the complexed phenol
and the complexed phenolate. The absorbance values of the sam-
ples at a selected wavelength k, were related to the absorption
coefficients of the four species eMk
;
ekMHþ
;
ekMꢁWP6 ekMHþꢁWP6, the total
;
The values of the equilibrium constants obtained from the
experimental spectra are presented in Table 1.
concentrations, c0M and cW0 P6, the pH, in addition to the respective
equilibrium constant. Such expressions were derived starting from
Beer’s law, the mass balance equations and the equations of the
equilibrium constants [36]. A detailed description of the methods
used for the determination of the equilibrium constants is pro-
vided in Section S2 of Supplementary Materials (SM).
3. Results and discussion
3.1. Synthesis of merocyanine dyes
The merocyanine dyes M1- M3 were synthesized as outlined in
Fig. 3. The synthesis of M1 (Brooker’s merocyanine) was performed
as described in Ref. [34]. The new compounds M2 and M3 were
prepared from 1-phenylpicolinium chloride [37] and 4-
hydroxybenzaldehyde or 3,5-dibromo-4-hydroxybenzaldehyde,
respectively. First, the phenol forms of the dyes were prepared
by the piperidine-catalyzed Knoevenagel condensation. The phe-
nolate forms (inner salts) were obtained then by treating the aque-
ous suspensions of the phenolate form dyes by NaOH.
Fig. 4. Scheme of reactions in merocyanine dye M – pillararene WP6 systems.
3

Dóra Hessz, S. Bádogos, Márton Bojtár et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 252 (2021) 119455
Table 1
The K3 equilibrium constants, corresponding to the protic equi-
libria of the complexed dyes, were determined from the pH depen-
dence of the spectra of samples containing WP6 in large excesses
over the initial concentrations of the dyes. As example, the pH
dependent spectra of the M3-WP6 system is shown in see
Fig. 5b. The variation of the spectra of all the three M-WP6 systems
with the pH can be seen in Fig. S11 in SM. The presence of isobestic
points in the spectra confirmed that the spectra were the linear
combinations of the spectra of two absorbing components, MꢁWP6
and MH+ꢁWP6, the contributions of the uncomplexed forms of the
dye, M and MH+ were negligible.
Equilibrium constants K1 – K4 for the reactions in merocyanine dye – WP6 mixtures.a
System
Acidic dissociation
constants
Binding constants
(Lmolꢂ1
)
K2
K4
ꢂlogK1
ꢂlogK3
10.23
9.51
5.99
M1 – WP6
M2 – WP6
M3 – WP6
8.66, (8.57)b
8.44
1.24ꢁ105
4.90ꢁ106
1.05ꢁ105
1.26ꢁ106
5.25 ꢁ107
1.66ꢁ106
4.79
a
This work, determined in Britton-Robinson buffers with ionic strength of
0.2 mol/L.
b
Ref. [31].
The spectral changes in the M1-WP6 and M2-WP6 systems
could be followed in the full range of the acid-base equilibrium,
allowing an accurate determination of the K3 equilibrium con-
stants, using K3 as the only fitting parameter. In case of the mix-
tures of the more acidic M3 with WP6, the pure protonated
complex could not be approached up to the pH limit of 6.0. In this
case, the absorption coefficients of the complex in the range of its
absorption band were also treated as fitting parameters.
Comparing the pKa values for the free and complexed merocya-
nines in Table 1, it can be seen that the acidity of all the three
merocyanines decreased substantially in the complexes. Presum-
ably, the increase of pKa is a combined effect of the reduced polar-
ity of the local environment of the dyes in the pillararene cavity
and the electrostatic field of the carboxylate groups of the WP6
3.2.2. Acidic dissociation constants
First, the dissociation constants K1 for the phenol forms of M1-
M3 were determined from their absorption spectra measured as
function of pH. The results are illustrated in Fig. 5a showing the
spectra of M3. (The pH dependent spectra of all the three merocya-
nine dyes are presented in Fig. S10 in SM). As can be seen in Table 1,
the pKa value of M2H+ was very close to the pKa of M1H+, the latter
reported also in Ref.[31] In contrast, with the two bromines in the
phenol part of M3, the desired enhancement of the acidity was
achieved – M3 was present in its zwitterionic form in neutral
solutions.
Fig. 5. pH dependent spectra of (a) 1.0ꢁ10ꢂ5 M solution of M3 and (b) a mixture of 1.0ꢁ10ꢂ5 mol/L M3 and 2.0ꢁ10ꢂ4 mol/L WP6. The red trace is the spectrum of complex
M3H+ꢁWP6 obtained by least-squares fitting. The insets show the plots of the absorbance values at the band maxima of (a) merocyanine M3 and (b) its WP6 complex versus
the pH of the samples.
Fig. 6. Absorption spectra of (a) M1-WP6 and (b) M3 – WP6 mixtures in pH 6.50 solutions. Total concentrations (a) c0M1 = 1.0∙10ꢂ5 mol/L, c0WP6 = 0ꢂ4∙10ꢂ5 mol/L; (b)
cM0 3 = 1.0∙10ꢂ5 mol/L, cW0 P6 = 0–5∙10ꢂ5 mol/L. The violet traces in (b) show the contributions of M3ꢁWP6 and M3H+ꢁWP6 to the spectra of the sample with cW0 P6 = 5∙10ꢂ5 mol/L.
The insets show the plots of the absorbance values at the band maxima of (a) M1H+ and (b) M3 versus the total concentrations of WP6.
4

Dóra Hessz, S. Bádogos, Márton Bojtár et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 252 (2021) 119455
host. The pKa shifts of acid-base indicators in micelles of charged
surfactants has been evaluated in terms of the above combined
effect [38,39].
were formed. In contrast, the phenolate form of the stronger acidic
dye, M3 formed only one type of complex, M3ꢁWP6 with the pil-
lararene host in these basic solutions, no proton-uptake was indi-
cated by the spectra in Fig. 7b.
The spectra of the M1-WP6 and M2-WP6 systems were also
measured in 0.2 M NaOH solutions (see Fig. S13). These spectra
were the linear combinations of the spectra of the phenolate form
dyes and their WP6 complexes, the bands of the of the phenol form
merocyanine – WP6 complexes were not observable in these
strongly basic solutions.
The spectral data of the dyes in the absence of WP6 and at high
WP6 concentrations where the complexation is close to complete,
are compared in Table 2. Both in the cases of the phenol and phe-
nolate form dyes, the complexation results in 20–40 nm shifts of
the absorption bands to higher wavelengths. The shifts of the zwit-
terionic (phenolate) forms are accordance with the lower local
polarity in the cavity of the WP6 host compared to the polarity
in bulk water. The visible band of M1 also shifts to the red in the
spectra of its cyclodextrin complexes [10,11,12]. The solva-
tochromism of the phenol forms of Brooker merocyanines have
not been analyzed yet in detail. A similar red shift has been
observed in the spectrum of DASPI (the pyridinium styryl dye with
a dimethylamino substituent in the place of the OH group of M1H+)
in the presence of WP6 [27].
3.2.3. Spectra of merocyanine dye – WP6 complexes
The complexation of the three merocyanine dyes were studied
on the spectra of mixtures with the same dye and different WP6
concentrations. First, two sets of spectra were recorded for each
dye-WP6 system, one set at pH 6.5 (see Fig. 6), the other set at
pH 10.5 (see Fig. 7). As mentioned above, the dissociation of WP6
can be taken complete only in pH > 6.0 solutions.
As follows from their pKa values, at pH 6.5 the uncomplexed M1
and M2 dyes are present dominantly in their undissociated phenol
form, whereas M3 is present in its phenolate form. Adding WP6 to
the solution of M1, a new band emerged at higher wavelength (see
Fig. 6a). This band belongs to the complexed phenol, M1H+ꢁWP6.
The spectra of the M2H+ - WP6 mixtures showed similar changes
(Fig. S12b).
The addition of WP6 to the pH 6.5 solution of M3 resulted in the
appearance of two new features (see the resolved spectrum in
Fig. 6b). A comparison of the spectra to the spectra of M3 at various
pH-s, in the presence of WP6 in large excess (Fig. 5b), made it clear
that the new bands belonged to the complexes of the phenolate
and phenol forms of the dye, M3ꢁWP6 and M3H+ꢁWP6. The forma-
tion of the phenol complex from the phenolate guest was an addi-
tional evidence that the acidity of M3 was reduced significantly on
the coordination by WP6.
The spectra of the M1-WP6 systems, measured at pH = 10.5 (see
Fig. 7a) contained also the contributions of three absorbing compo-
nents, like the spectra of M3-WP6 systems measured at pH 6.5 (see
Fig. 6b). These spectra proved that the reaction of the deprotonated
dye, M1 with WP6 yielded a mixture of the complexes with proto-
nated and deprotonated dye guests, M1H+ꢁWP6 and M1ꢁWP6. Sim-
ilarly, in the M2 – WP6 mixtures both M2H+ꢁWP6 and M2ꢁWP6
3.2.4. Binding constants
The binding constants K2 were determined by a one parameter
fitting to the spectra of the three dyes in basic solutions, in the
presence of WP6 in different concentrations. In case of M3ꢁWP6,
the spectra measured in pH 10.5 buffers (Fig. 7b) were used as
experimental data. In cases of the complexes M1ꢁWP6 and
M2ꢁWP6, the pKa-s of which fall in the basic range, the values of
K2 were obtained from spectra measured in 0.2 mol/L NaOH solu-
tions (see Figs. S13a-b in the SM).
Fig. 7. Absorption spectra of (a) M1-WP6 and (b) M3 – WP6 mixtures in pH 10.5 solutions. Total concentrations are (a) c0M1 = 1.0ꢁ10ꢂ5 M, cW0 P6 = 0–1.2ꢁ10ꢂ4 mol/L; (b)
cM0 3 = 1.0ꢁ10ꢂ5 mol/L, cW0 P6 = 0–1.2ꢁ10ꢂ4 mol/L. The violet traces in (a) show the contributions of M1ꢁWP6 and M1H+ꢁWP6 to the spectra of the sample with cW0 P6 = 1.2ꢁ10ꢂ4 mol/
L. The insets show the plots of the absorbance values at the band maxima of (a) M1 and (b) M3 versus the total concentration of WP6.
Table 2
Absorption spectral data of neutral and protonated forms of merocyanine dyes M1-M3 and their WP6 complexes.
MH+
M
MH+ꢁWP6
MꢁWP6
kmax
[nm]
e
kmax
[nm]
e
kmax
[nm]
e
kmax
[nm]
e
[Lmolꢂ1cmꢂ1
]
[Lmolꢂ1cmꢂ1
]
[Lmolꢂ1cmꢂ1
]
[Lmolꢂ1cmꢂ1
]
M1
M2
M3
374
394
377
36,500
35,500
36,700
444
475
472
42,800
35,600
37,000
396
415
394
26,700
29,500
33,100
479
506
495
36,500
30,500
37,100
5

Dóra Hessz, S. Bádogos, Márton Bojtár et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 252 (2021) 119455
Fig. 8. Spectral changes of a M3–WP6 mixture (c0M3 = 2ꢁ10ꢂ5 mol/L, cW0 P6 = 6ꢁ10ꢂ5 mol/L) on the addition of Lys and its methylated derivatives. (a) Absorbance values at
452 nm, (b) photographs of solutions of M3, M3 + WP6 and M3 + WP6 + Me3Lys (c0M3 = 2ꢁ10ꢂ5 mol/L, c0WP6 = 6ꢁ10ꢂ5 mol/L, cM0 e
= 2.2ꢁ10ꢂ3 mol/L).
3Lys
The values of K4 for M1 and M2 were obtained in a similar man-
ner, from the spectra of dye-WP6 mixtures with high WP6
excesses, measured in pH 6.5 buffers (Fig. 6a and S12b, the latter
in the SM). In case of the stronger acidic dye M3, the binding con-
stant K4 could not be determined directly, since the undissociated
pyridinium phenol form, M3H+ is dominant only at low pH values
where WP6 is not fully dissociated. The value of K4 for M3 was esti-
mated as K4 = (K1ꢁK2)/K3.
The colorimetric sensor in the displacement experiments was
an M3-WP6 mixture with initial concentrations c0M3 = 2ꢁ10ꢂ5
mol/L and c0WP6 = 6ꢁ10ꢂ5 mol/L. The pH was set to 8.0. Calculated
from the K2 binding constant, 86% of the indicator was in com-
plexed state in this mixture.
The variations of the spectra with the concentrations of Lys and
its methylated derivatives are illustrated in Fig. 8. As can be seen in
the figure, Me3Lys induced a strong change in the spectrum, the
color change was visible even by naked eye. Me2Lys induced a
weaker spectral response, whereas the effects of MeLys and Lys
on the spectra were negligible.
Comparing the binding constants K2 and K4 in Table 1, it can be
seen that the value of K4 is higher by one order magnitude than the
value of K2 for the same dye. These differences are due to the
strong electrostatic interactions between the positively charged
MH+ guests and the negatively charged WP6 host. The electrostatic
interactions between the dye zwitterions and the WP6 host are
weaker.
The spectra of the M3 – WP6 – MenLys mixtures afforded the
calculation of the binding constants for the WP6 complexes of
Me3Lys and Me2Lys. The compositions of the displacement assays
were governed by the simultaneous reactions
The highest K2 value as well as the highest K4 value belong to
the complexes of M2. Presumably, the phenyl group on the pyri-
dinium N atom intrudes deeply into the cavity of the WP6 hosts
of the M2ꢁWP6 and M2H+ꢁWP6 complexes. This way, these com-
plexes are also stabilized by a strong hydrophobic effect.
Hydrophobic interactions must also contribute significantly to
the stabilization of the complexes M3ꢁWP6 and M3H+ꢁWP6 since
the pyridinium group of M3 is also supplied with a phenyl sub-
stituent. The values of the K2 and K4 binding constants of the
M3-WP6 complexes are lower than the respective values of the
M2-WP6 complexes. This difference may arise from the better sol-
vation of the dibrominated phenol moiety of M3 by water solvent
molecules.
M þ WP6¢M ꢁ WP6; with equilibrium constant K2
and
A þ WP6¢A ꢁ WP6; with equilibrium constant K5;
where A denotes the Me3Lys or Me2Lys analyte.
The value of K2 was obtained earlier from the spectra of binary
M3 –WP6 systems. A fitting to the spectra of the M3 –WP6 – Me3-
Lys and M3 –WP6 – Me2Lys ternary systems yielded K5 = 2.7 ∙ 103
Lmolꢂ1 for Me3LysꢁWP6 and 6.6 ∙ 102 Lmolꢂ1 for Me2LysꢁWP6. It is
worth to note that WP5, the smaller ring-sized analogue of WP6,
forms a complex of higher stability with Lys than with Me3Lys
[40]. This reverse order of the affinities is an interesting example
of size-selective complexation in supramolecular chemistry.
3.3. Indicator displacement
4. Conclusions
The phenol and the phenolate forms of the three merocyanines
showed similar spectral shifts on complexation, the absorption
band of the complexes were located at ~30 nm higher wavelengths
than the bands of the uncomplexed guests. The advantages of the
usage of a phenolate form dye as indicator for a displacement assay
were that (i) the phenolates and their WP6 complexes absorbed in
the visible range, thus, the complexation could be detected by
naked eyes; (ii) their binding constants (K2) were lower, than the
binding constants of the phenol forms (K4), making the displace-
ment more efficient. The indicator M3 had the additional advan-
tage of its low pKa, the phenolate forms of the free and
complexed indicator were dominant in neutral solutions. In con-
trast, in case of M1 and M2 this condition was met only in strongly
basic media.
Our work on the complexes of the pyridinium phenolate dyes
M1-M3 with carboxylato pillar[6]arene WP6 showed that the dyes
occur in four forms in the systems which are the undissociated
cationic phenol, MH+ and its complex, MH+ꢁWP6 and the zwitteri-
onic phenolate form, M and its complex, MꢁWP6. The two acidic
dissociation constants and the two binding constants governing
the compositions of the systems were determined from the
absorption spectra. The absorption bands of the zwitterionic forms
showed a large batochromic shift on complexation, making M1-M3
suitable indicators for WP6-based indicator displacement systems.
Comparing the pKa-s and the binding constants of the three dyes,
the dibromo derivative M3 was selected as an indicator of such
sensing system for the detection of cationic biomolecules. The
6

Dóra Hessz, S. Bádogos, Márton Bojtár et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 252 (2021) 119455
[13] J.S. Holt, Structural characterization of the Brooker’s merocyanine/b-
cyclodextrin complex using NMR spectroscopy and molecular modeling, J.
Mol. Struct. 965 (2010) 31–38.
[14] Y.-T. Kang, X.-Y. Tang, H.-D. Yu, Z.-G. Cai, Z.-H. Huang, D. Wang, J.-F. Xu, X.
Zhang, Supramolecular catalyst functions in catalytic amount: cucurbit[8]uril
accelerates the photodimerization of Brooker’s merocyanine, Chem. Sci. 8
(2017) 8357–8361.
M3-WP6 complex was found an effective assay for the detection of
Me3Lys. As the pKa values of pyridinium phenolate dyes increase
much on complexation, the binding of the free phenol forms of
M1-M3 and analogous dyes to WP6 leads to the complexes of
the phenolate forms in the appropriate pH interval, providing fur-
ther perspectives for the design of pillararene-based
chemosensors.
[15] Y.-T. Kang, Z.-G. Cai, Z.-H. Huang, X.-Y. Tang, J.-F. Xu, X. Zhang, Controllable
Supramolecular
Polymerization
Promoted
by
Host-Enhanced
Photodimerization, ACS Macro Lett. 5 (2016) 1397–1401.
[16] M.M. Linn, D.C. Poncio, V.G. Machado, An anionic chromogenic sensor based
on the competition between the anion and a merocyanine solvatochromic
dye for calix[4]pyrrole as a receptor site, Tetrahedron. Lett. 48 (2007) 4547–
4551.
[17] G.C. Yu, X.R. Zhou, Z.B. Zhang, H.Y. Han, Z.W. Mao, C.Y. Gao, F.H. Huang, Pillar
[6]arene/paraquat molecular recognition in water: high binding strength, pH-
responsiveness, and application in controllable self-assembly, controlled
release, and treatment of paraquat poisoning, J. Am. Chem. Soc. 134 (2012)
19489–19497.
[18] Y. Cao, X.-Y. Hu, Y. Li, X.-C. Zou, S.H. Xiong, C. Lin, Y.-Z. Shen, L.Y. Wang,
Multistimuli-responsive supramolecular vesicles based on water- soluble
Pillar[6]arene and SAINT complexation for controllable drug release, J. Am.
Chem. Soc. 136 (2014) 10762–10769.
[19] Q.Z. Zhou, H.J. Jiang, R.N. Chen, F.L. Qiu, G.L. Dai, D.M. Han, triply-responsive
pillar[6]arene-based supramolecular amphiphile for tunable formation of
vesicles and controlled release, Chem. Commun. 50 (2014) 10658–10660.
[20] X.D. Chi, P. Wang, Y. Li, X.F. Ji, Thermo-triggered release of a Cys probe from
the cavity of a water-soluble pillar[5]arene, Tetrahedron. Lett. 56 (2015) 4545–
4548.
CRediT authorship contribution statement
Dóra Hessz: Investigation, Methodology, Formal analysis. Stella
Bádogos: Investigation. Márton Bojtár: Investigation, Writing -
original draft. István Bitter: Conceptualization. László Drahos:
Investigation. Miklós Kubinyi: Project administration, Funding
acquisition, Conceptualization, Writing - original draft, Writing -
review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
[21] M. Bojtár, Z. Szakács, D. Hessz, M. Kubinyi, I. Bitter, Optical spectroscopic
studies on the complexationof stilbazolium dyes with a water soluble pillar
[5]-arene, RSC Adv. 5 (2015) 26504–26508.
Acknowledgement
[22] M. Bojtár, A. Paudics, D. Hessz, M. Kubinyi, I. Bitter, Amino acid recognition
by fine tuning the association constants: tailored naphthalimides in pillar
The research reported in this paper and carried out at BME has
been supported by the NRDI Fund (TKP2020 IES, Grant BME-IE-
BIO) based on the charter of bolster issued by the NRDI Office
under the auspices of the Hungarian Ministry for Innovation and
Technology.
[5]arene-based indicator displacement assays, RSC Adv.
86275.
6 (2016) 86269–
[23] X.-D. Xiao, L. Shi, L.-H. Guo, J.-W. Wang, X. Zhang, Determination of dopamine
hydrochloride by host-guest interaction based on water-soluble pillar[5]arene,
Spectrochim. Acta A 173 (2017) 6–12.
[24] L. Barbera, D. Franco, L.M. De Plano, G. Gattuso, S.P.P. Guglielmino, G. Lentini,
N. Manganaro, N. Marino, A water-soluble pillar[5]arene as a new carrier for
an old drug, Org. Biomol. Chem. 15 (2017) 3192–3195.
[25] B. Hua, L. Shao, Z.H. Zhang, J.F. Sun, J. Yang, Pillar[6]arene/acridine orange host-
guest complexes as colorimetric and fluorescence sensors for choline
compounds and further application in monitoring enzymatic reactions, Sens.
Actuat. B 255 (2018) 1430–1435.
[26] K. Yang, J. Wen, S. Chao, J. Liu, K. Yang, Y.X. Pei, Z.C. Pei, A supramolecular
photosensitizer system based on the host-guest complexation between water-
soluble pillar[6]arene and methylene blue for durable photodynamic therapy,
Chem. Commun. 54 (2018) 5911–5914.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.119455.
References
[27] A. Paudics, M. Kubinyi, I. Bitter, M. Bojtár, Carboxylato-pillar[6]arene-based
fluorescent indicator displacement assays for the recognition of monoamine
neurotransmitters, RSC Adv. 9 (2019) 16856–16862.
[28] S. Chao, X.K. Lv, N. Ma, Z.Y. Shen, F.Y. Zhang, Y.X. Pei, Z.C. Pei, A supramolecular
nanoprodrug based on a boronate ester linked curcumin complexing with
water-soluble pillar[5]arene for synergistic chemotherapies, Chem. Commun
56 (2020) 8861–8864.
[29] L. Shao, Y.T. Pan, B. Hua, S.D. Xu, G.C. Yu, M.B. Wang, B. Liu, F.H. Huang,
Constructing Adaptive Photosensitizers via Supramolecular Modification
Based on Pillararene Host-Guest Interactions, Angew. Chem. Int. Ed. 59
(2020) 11779–11783.
[30] R. Varshney, M. Alam, C. Agashe, R. Joseph, D. Patra, Pillar[5]arene
microcapsules turn on fluid flow in the presence of paraquat, Chem.
Commun. 56 (2020) 9284–9287.
[31] J.E. Kuder, D. Wychick, Acid-base equilibria in the ground and excited states of
a solvatochromic merocyanine dye, Chem. Phys. Lett. 24 (1974) 69–72.
[32] C.T. Martins, M.S. Lima, O.A. El Seoud, Thermosolvatochromism of
merocyanine polarity indicators in pure and aqueous solvents: relevance of
solvent lipophilicity, J. Org. Chem. 71 (2006) 9068–9079.
[33] G.C. Yu, M. Xue, Z.B. Zhang, J.Y. Li, C.Y. Han, F.H. Huang, A water-soluble Pillar
[6]arene: synthesis, host-guest chemistry, and its application in dispersion of
multiwalled carbon nanotubes in water, J. Am. Chem. Soc. 134 (2012) 13248–
13251.
[34] T. Kolev, B.B. Koleva, S. Stoyanov, M. Spiteller, I. Petkov, The aggregation of the
merocyanine dyes, depending of the type of the counterions, Spectrochim.
Acta A 70 (2008) 1087–1096.
[35] C. Monday, V. Cerda, A Britton-Robinson buffer of known ionic strength, Ann.
Chim. 64 (1974) 409–412.
[1] L.G.S. Brooker, G.H. Keyes, D.W. Heseltine, Color and constitution. XI.
anhydronium bases of p-hydroxystyryl dyes as solvent polarity indicators, J.
Am. Chem. Soc. 73 (1951) 5350–5356.
[2] P. Jacques, On the relative contributions of nonspecific and specific
interactions to the unusual solvatochromism of a typical merocyanine dye, J.
Phys. Chem. 90 (1986) 5535–5539.
[3] M. Dominguez, M.C. Rezende, Towards a unified view of the solvatochromism
of phenolate betaine dyes, J. Phys. Org. Chem. 23 (2010) 156–170.
[4] N.A. Murugan, J. Kongsted, Z. Rinkevicius, K. Aidas, H. Agren, Modeling the
structure and absorption spectra of stilbazolium merocyanine in polar and
nonpolar solvents using hybrid QM/MM techniques, J. Phys. Chem. B 114
(2010) 13349–13357.
[5] N.A. Murugan, J. Kongsted, Z. Rinkevicius, H. Agren, Demystifying the
solvatochromic reversal in Brooker’s merocyanine dye, Phys. Chem. Chem.
Phys. 13 (2011) 1290–1292.
[6] V. Manzoni, K. Coutinho, S. Canuto, An insightful approach for understanding
solvatochromic reversal, Chem. Phys. Lett. 655–656 (2016) 30–34.
[7] T. Bevilaqua, D.C. da Silva, V.G. Machado, Preferential solvation of Brooker’s
merocyanine in binary solvent mixtures composed of formamides and
hydroxylic solvents, Spectrochim. Acta A 60 (2004) 951–958.
[8] F.M. Testoni, E.A. Ribeiro, L.A. Giusti, V.G. Machado, Merocyanine
solvatochromic dyes in the study of synergistic effects in mixtures of
chloroform with hydrogen-bond accepting solvents, Spectrochim. Acta A 71
(2009) 1704–1711.
[9] E.A. Ribeiro, T. Sidooski, L.G. Nandi, V.G. Machado, Interaction of protonated
merocyanine dyes with amines in organic solvents, Spectrochim. Acta A 81
(2011) 745–753.
[10] C. de Garcia Venturini, J. Andreaus, V.G. Machado, C. Machado, Solvent effects
in the interaction of methyl-b-cyclodextrin with solvatochromic merocyanine
dyes, Org. Biomol. Chem. 3 (2005) 1751–1756.
[36] A.E. Hargrove, Z.L. Zhong, J.L. Sessler, E.V. Anslyn, Algorithms for the
determination of binding constants and enantiomeric excess in complex
host : guest equilibria using optical measurements, New J. Chem. 34 (2010)
348–354.
[37] B.J. Coe, J.A. Harris, I. Asselberghs, A. Persoons, J.C. Jeffery, L.H. Rees, T. Gelbrich,
M.B. Hursthouse, Tuning of charge-transfer absorption and molecular
quadratic non-linear optical properties in ruthenium(II) ammine complexes,
J. Chem. Soc., Dalton Trans. (1999) 3617–3625.
[11] J. Nicolini, C.G. Venturini, J. Andreaus, C. Machado, V.G. Machado, Interaction of
Cyclodextrins with Brooker’s Merocyanine in Aqueous Solution, Spectrosc.
Lett. 42 (2009) 35–41.
[12] J.S. Holt, A. Campitella, A. Rich, J.L. Young, Spectroscopic characterization of the
binding and isomerization cycle of Brooker’s merocyanine with
a-, b- and c-
cyclodextrins, J. Inc., Phenom. Macorocycl. Chem. 61 (2008) 251–258.
7

Dóra Hessz, S. Bádogos, Márton Bojtár et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 252 (2021) 119455
[38] N.O. Mchedlov-Petrossyan, Protolytic equilibrium in lyophilic nanosized
dispersions: Differentiating influence of the pseudophase and salt effects,
Pure Appl. Chem. 80 (2008) 1459–1510.
[39] N.O. Mchedlov-Petrossyan, V.S. Farafonov, T.A. Cheipesh, S.V. Shekhovtsov, D.
A. Nerukh, A.V. Lebed, In search of an optimal acid-base indicator for
examining surfactant micelles: Spectrophotometric studies and molecular
dynamics simulations, Colloids Surf. A 565 (2019) 97–107.
[40] C.-J. Li, J.-W. Ma, L. Zhao, Y.-Y. Zhang, Y.-H. Yu, X.-Y. Shu, J. Lia, X.-S. Jia,
Molecular selective binding of basic amino acids by a water-soluble pillar[5]
arene, Chem. Commun. 49 (2013) 1924–1926.
8