Benzothiazole merocyanine dyes as middle pH optical sensors

Article abstract of DOI:10.1016/j.dyepig.2020.108193

Substituted merocyanine dyes were obtained by Knoevenagel condensation between formylated arylbenzothiazole and quaternary 2-alkylbenzothiazoles. The condensations were performed in the presence of sodium methoxide and the products were obtained with exce

Full text of DOI:10.1016/j.dyepig.2020.108193

Journal Pre-proof
Benzothiazole merocyanine dyes as middle pH optical sensors
Felipe Lange Coelho, Rodrigo da Costa Duarte, Cláudia de Ávila Braga, Josene
Maria Toldo, Paulo Fernando Bruno Gonçalves, Fabiano da Silveira Santos, Fabiano
Severo Rodembusch
PII:
S0143-7208(19)32907-9
DOI:
https://doi.org/10.1016/j.dyepig.2020.108193
DYPI 108193
Reference:
To appear in: Dyes and Pigments
Received Date: 11 December 2019
Revised Date: 7 January 2020
Accepted Date: 7 January 2020
Please cite this article as: Coelho FL, da Costa Duarte R, de Ávila Braga Clá, Toldo JM, Bruno
Gonçalves PF, da Silveira Santos F, Rodembusch FS, Benzothiazole merocyanine dyes as middle pH
optical sensors, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108193.
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Benzothiazole merocyanine dyes as middle pH optical sensors
Author: Felipe Lange Coelho1,†
Synthesis and spectroscopic characterization
Author: Rodrigo da Costa Duarte1,†
Synthesis and spectroscopic characterization
Author: Cláudia de Ávila Braga1
Synthesis and experimental photophysics
Author: Josene Maria Toldo2
Theoretical calculations
Author: Paulo Fernando Bruno Gonçalves2
Theoretical calculations
Author: Fabiano da Silveira Santos1,*
Experimental photophysics and data treatment
Author: Fabiano Severo Rodembusch1,*
Corresponding author. Revision of all data. Discussion of the results.
manuscript preparation.
1Grupo de Pesquisa em Fotoquímica Orgânica Aplicada, Universidade Federal
do Rio Grande do Sul/UFRGS. Av. Bento Gonçalves 9500. CEP 91501-970
Porto Alegre-RS, Brazil. E-mail: rodembusch@iq.ufrgs.br and fabiano@ufrgs.br
2Grupo de Química Teórica, Universidade Federal do Rio Grande do Sul –
Instituto de Química, Av. Bento Gonçalves 9500, CEP 91501-970, Porto Alegre,
RS, Brazil.
†The authors contributed equally to this work
1

Benzothiazole merocyanine dyes as middle pH optical sensors
Felipe Lange Coelho,1,† Rodrigo da Costa Duarte,1,† Cláudia de Ávila Braga,1
Josene Maria Toldo,2 Paulo Fernando Bruno Gonçalves,2 Fabiano da Silveira
Santos,1,* Fabiano Severo Rodembusch,1,*
1Grupo de Pesquisa em Fotoquímica Orgânica Aplicada, Universidade Federal
do Rio Grande do Sul/UFRGS. Av. Bento Gonçalves 9500. CEP 91501-970
Porto Alegre-RS, Brazil. E-mail: rodembusch@iq.ufrgs.br and fabiano@ufrgs.br
2Grupo de Química Teórica, Universidade Federal do Rio Grande do Sul –
Instituto de Química, Av. Bento Gonçalves 9500, CEP 91501-970, Porto Alegre,
RS, Brazil.
†The authors contributed equally to this work
Abstract
Substituted merocyanine dyes were obtained by Knoevenagel condensation
between formylated arylbenzothiazole and quaternary 2-alkylbenzothiazoles.
The condensations were performed in the presence of sodium methoxide and
the products were obtained with excellent yields after simple purification. The
merocyanines presented absorption maxima in the range 576-605 nm (green to
orange-red region). The calculated Strickler-Berg parameters indicated fully
1
spin and symmetry allowed electronic transitions with π-π* character. These
compounds show significant fluorescence emission at the orange red region
with a very small Stokes shift. Surprisingly, although the benzothiazole core
possesses a structural moiety (o-hydroxyaryl moiety) which should facilitate
proton transfer in the excited state, no evidence was found of this
phototautomerism in the compounds studied. TD-DFT calculations show that
ESIPT is not probable to happen due to the high barriers to transfer the proton.
In addition, the aliphatic chain does not affect the absorption and emission
maxima location. For both, ground and excited states, a negative
solvatochromic effect was observed. Theoretical calculations also show that this
effect is due to the larger dipole moment in the S0 than in the S1 state. In
addition, these compounds presented large shifts on the absorption spectra
1

changing the pH (yellow to violet color), which allowed studying successfully as
optical sensor for middle pH values.
Keywords: ESIPT, optical sensor, benzothiazole, merocyanine, colorimetric
sensor
1. Introduction
Merocyanines can be classified as polymethine dyes that are
characterized by neutral unsymmetrical structures composed of a nitrogen
donor and an oxygen acceptor moieties linked by one or more ethylene group
[1]. These compounds present photophysical and photochemical properties
such as pronounced solvatochromism, high photostability and nonlinear optical
and electrical responses [2]. These properties can be related to the charge
transfer from donor to acceptor fragments through the polymethine chain and
are modulated depending on the substitution of end and lateral groups [2]. In
this sense, several applications can be envisaged for these structures, including
optical data storage [3], solvatochromic sensor [4], biological markers [5-8],
photodynamic therapy [9-11] and solar cell devices (Figure 1) [12,13].
Figure 1. Selected examples of merocyanine derivatives and their main
applications presented in the literature and the merocyanines presented in this
work.
2

It can be found in the literature that the synthetical approaches to
merocyanine vary depending on the number of methylene groups in the
polymethine chain [14]. The general synthetic protocol to merocarbocyanines (n
= 1) involves Knoevenagel condensation between heterocyclic enamines or
their corresponding salts with substituted aldehydes [15], while the preparation
of simple merocyanines (n = 0) is mainly performed through condensation of
nitrogen heterocycles containing leaving groups in the 2- or 4-positon with
activated methylene group [16,17]. In case of meropolycarbocyanines (n > 1),
similar methods are used according to structural feature and reactivity of the
reactants [18,19].
In general, synthetic protocols for these dyes are robust and side
reactions and byproducts formation are not reported. Moreover, the broad
accessibility of starting materials, as well as the easy isolation of products, in
contrast with polymethine chain analogues (meropolycarbocyanines) become
their obtention more attractive [1,2].
For specific application as optical sensor for pH, the acidity constant
plays a fundamental role, where the pKa value is a fundamental parameter to
predict the ionization state concerning the pH [20-22]. However, organic dyes
are poorly water-soluble compounds. In order to overcome this drawback, that
difficult the pKa determination, mixtures of aqueous-organic solvents are used.
In this methodology, it is reported in the literature polar protic cosolvents [23-
25], as well as aprotic media, such as dimethylsulfoxide [26], N,N-
dimethylformamide [27], or acetonitrile [28]. In this sense, ethanol/water
solutions usually 1:1 (v/v) presents very good results for poorly water-soluble
compounds [27,29,30], where the cosolvent ionization constant (pKa) are
measured and the ionization constant in water (wpKa) is obtained by
extrapolation [31-34].
In this way, this investigation focus on the synthesis and photophysical
characterization in solution of merocyanine dyes containing a proton-transfer
moiety aiming an optical sensing application allowed by the chemical structure
of these compounds. In addition, their photophysics in the ground state was
evaluated against pH in order to obtain their ionization constants, as well as to
explore their ability as optical sensors for pH. Theoretical calculations were also
3

applied to better understand their photophysics and also the unprecedented
absence of proton transfer reaction in the excited state.
2. Experimental Section
2.1 Materials and methods
Purification by column chromatography was carried out on silica gel 60
(230−400 mesh). Analytical thin-layer chromatography (TLC) was conducted on
aluminum plates with 0.2 mm of silica gel 60F-254. The solvents were purified
using standard procedures [35] or used as received. Spectroscopic grade
solvents (Merck or Aldrich) were used for the photophysical characterization
(UV-Vis absorption and fluorescence emission). FTIR spectra were recorded on
13
a Varian 640-IR using KBr disc or at ATR mode. 1H (400 MHz) and C (100
MHz) NMR were obtained in CDCl3 or DMSO-d6 on a Bruker Avance III NMR
spectrometer. The chemical shifts are expressed as δ (ppm) relative to
tetramethylsilane (TMS). Splitting patterns are designed as follows; s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br s, broad singlet. All of the detected
signals were in accordance with the proposed structures. UV-Vis absorption
spectra were obtained using a Shimadzu UV-2450 spectrophotometer. Steady-
state fluorescence spectra were measured using
a
Shimadzu
spectrofluorometer model RF-5301PC. Fluorescence quantum yields (QY) were
measured at 25°C using solutions at the optical dilute regime and using Quinine
sulfate in H2SO4 1 N as the quantum yield standards [36]. The ionization
constant was obtained in accordance with the literature [28]. The pH values
were measured on a Digimed DM-22 pH-meter with flow-through glass-silver
chloride electrodes filled with a KCl saturated solution. The ionization constants
were determined by UV-Vis absorption spectroscopy [23,33,34,29,30,37]. The
UV-Vis titrations were performed with constant ionic strength of the solutions
(µ=0.1) by the addition of KCl (0.15 M) under nitrogen atmosphere at 25°C [38].
The measurements were performed in EtOH/H2O mixtures (1:1 v/v) with
different pH values. The initial solution was acidified using HCl (1 Molar) up to
pH 0.5 followed by additions of different amounts of KOH (1 Molar) up to pH 14.
Stock solutions of the studied in methanol (~10-6M) were prepared. Different
amounts of these solutions were added to the previous ethanol/water mixture at
different pH. The calculations were carried out minimizing the sum of squared
4

residuals of the absorbance values as a function of the wavelength under
different pH conditions by Boltzmann Function. The pH value vs. absorbance
data was adjusted with good correlation coefficients, where the inflection point
indicates where the pH=pKa in the Henderson-Hasselbach equation [39].
2.2 Synthesis and characterization
2-(2’-hydroxy-3’-methylphenyl)benzothiazole (3)
In a round-bottom vessel 3-methylsalicylic acid (2) (5.5 mmol) and
aminothiophenol (1) (5.0 mmol) were added. Then polyphosphoric acid (12 mL)
was added and the viscous mixture was stirred at 120ºC for 2 h. The
temperature was increased to 170ºC and stirring was continued for another 3 h.
After this, the reaction mixture was cooled and crude poured into ice-cold water
and neutralized with NaHCO3. The precipitate was filtered and crude solid was
dried prior to purification by column chromatography using AcOEt/hexane (5:95)
as eluent. The desired product was obtained as a white solid with 62% yield
(0.364 g). M.p. = 134-136 ºC. 1H NMR (400 MHz, CDCl3, δ ppm) 12.77 (s, 1H),
7.95 (d, J=8.05 Hz, 1H), 7.87 (d, J=7.97 Hz, 1H), 7.53 (d, J=7.88 Hz, 1H), 7.48
(dt, J=7.2, 1.2 Hz, 1H), 7.38 (dt, J=7.2, 1.1 Hz, 1H), 7.26-7.22 (m, 1H), 6.85 (t,
13
J=7.6 Hz, 1H), 2.35 (s, 3H). C NMR (100 MHz, CDCl3), (400 MHz, CDCl3, δ
ppm) 169.8, 156.3, 151.8, 133.7, 132.7, 126.9, 126.6, 125.4, 122.0, 121.5,
119.0, 116.0, 16.0. FTIR (ATR, ν = cm-1) 1618, 1260. HRMS (ESI/Q-TOF) m/z:
[M + H]+ calcd for C14H12NOS 242.0640; found 242.0635.
2-(5’-formyl-2’-hydroxy-3’-methylphenyl)benzothiazole (4)
In
a
Schlenk
vessel,
compound
3
(0.15
mmol)
and
hexamethylenetetramine (10 eq.) were added followed by the addition of
trifluoroacetic acid (2.5 mL). The mixture was heated at reflux for 5 h. The
progress of the chemical reaction was monitored by TLC. Water (2.5 mL) was
added and the mixture was refluxed for 60 min. The reaction mixture was kept
overnight at 5ºC. The obtained precipitate was filtered in silica, washed with
water, extracted in ethyl acetate affording desired product as a pale yellow solid
with 84% yield (0.038 g). M.p. = 180-186°C. 1H NMR (400 MHz, CDCl3, δ ppm)
13.42 (s, 1H), 9.78 (s, 1H), 7.90-7.81 (m, 3H), 7.63 (s, 1H), 7.48-7.34 (m, 2H),
5

13
2.32 (s, 3H). C NMR (100 MHz, CDCl3, δ ppm) 189.9, 168.4, 161.3, 151.0,
133.7, 132.4, 128.6, 128.0, 127.9, 126.7, 125.8, 122.0, 121.4, 115.9, 15.9. FTIR
(ATR, ν = cm-1) 1687, 1610, 1273. HRMS (ESI/Q-TOF) m/z: [M + H]+ calcd for
C15H12NO2S 270.0589; found 270.0587.
2.3 General protocol for quaternary indoles 6-7
To degassed N,N-dimethylformamide (15 mL) was added 2-
methylbenzo[d]thiazole (0.20 g, 1.34 mmol) and respective alkyl iodide (8.0
mmol) under N2 atmosphere. The reaction mixture was stirred at 55 ºC for 24h.
Crude was diluted with ethyl acetate (50 mL) and the precipitate was filtered
and washed with hot ethyl acetate (100 mL).
2,3-dimethylbenzo[d]thiazol-3-ium iodide (6)
1
Yield: 90 %. H NMR (400 MHz, DMSO-d6, δ ppm) 8.45 (d, J=8.4 Hz,
1H), 8.27 (d, J=8.4 Hz, 1H), 7.85 (t, J=8.5 Hz, 1H), 7.76 (t, J=8.5 Hz, 1H), 4.20
13
(s, 3H), 3.19 (s, 3H). C NMR (100 MHz, CDCl3, δ ppm) 177.5, 142.0, 129.7,
129.1, 128.5, 125.0, 117.2, 37.1, 18.1.
2-methyl-3-octylbenzo[d]thiazol-3-ium iodide (7)
1
Yield: 44 %. H NMR (400 MHz, CDCl3, δ ppm) 8.49 (d, J=8.2 Hz, 1H),
8.10 (d, J=8.5 Hz, 1H), 7.82 (t, J=8.5 Hz, 1H), 7.72 (t, J=8.5 Hz, 1H), 4.85 (d,
J=9.9 Hz, 2H), 3.44 (s, 3H), 2.05-1.88 (m, 2H), 1.50 (m, 2H), 1.37-1.19 (m, 8H),
0.91-0.79 (m, 3H). 13C NMR (100 MHz, CDCl3, δ ppm) 174.6, 140.8, 130.0,
129.0, 128.6, 125.0, 116.5, 51.3, 31.6, 29.0, 28.9, 28.7, 26.7, 22.4, 19.8, 14.0.
2.4 General procedure for merocyanine dyes 8-9
A solution of 4 (0.05 g, 0.18 mmol) in ethanol (10 mL) was added
dropwise to a previously prepared solution of corresponding quaternary indole
6-8 (0.22 mmol) and sodium methoxide (0.011 g, 0.20 mmol) in ethanol (5 mL).
The reaction mixture was stirred under reflux for 24h. Desired product
precipitate as a fine powder and was centrifuged and washed with cold ethanol.
No additional purification was performed.
6

(E)-2-(3-(benzo[d]thiazol-2-yl)-4-hydroxy-5-methylstyryl)-3-
methylbenzo[d]thiazol-3-ium iodide (8)
Yield: 97%. 1H NMR (400 MHz, DMSO-d6/TFA, δ ppm) 8.38 (d, J=7.4 Hz,
2H), 8.34 (s, 1H), 8.26 (d, J=15.8 Hz, 1H), 8.19 (m, 2H), 8.09 (d, J=7.9 Hz, 1H),
7.91 (d, J=15.8 Hz, 1H), 7.83 (m, 1H), 7.73 (t, J=7.2 Hz, 1H), 7.56 (m, 1H), 7.48
(m, 2H), 4.34 (s, 3H), 2.35 (s, 3H). 13C NMR (100 MHz, DMSO-d6/TFA, δ ppm)
172.4, 168.2, 159.5, 151.2, 148.5, 142.4, 133.6, 133.1, 130.5, 129.7, 128.6,
128.13, 128.09, 127.5, 126.4, 126.1, 124.5, 122.7, 122.3, 117.1, 117.0, 112.1,
+
36.6, 16.2. HRMS (ESI) calcd. for C24H19N2OS2 (M+) 415.0939, found
415.0936.
(E)-2-(3-(benzo[d]thiazol-2-yl)-4-hydroxy-5-methylstyryl)-3-octylbenzo[d]thiazol-
3-ium iodide (9)
Yield: 78%. 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.45 (d, J=8.1 Hz, 1H),
8.38 (s, 1H), 8.33 (d, J=15.7 Hz, 1H), 8.28 (d, J=8.5 Hz, 1H), 8.28 (m, 2H), 8.12
(d, J=5.4 Hz, 1H), 7.96-7.86 (m, 2H), 7.78 (t, J=11.7 Hz, 1H), 7.6 (td, J=5.0, 3.7
Hz, 1H), 7.51 (m, 1H), 4.96 (t, J=7.2 Hz, 2H), 2.38 (s, 3H), 1.86 (m, 2H), 1.41
13
(m, 2H), 1.32 (m, 2H), 1.20 (s, 6H), 0.78 (t, J = 6.71 Hz, 3H). C NMR (100
MHz, DMSO-d6, δ ppm) 172.2, 168.1, 151.2, 149.0, 141.6, 133.5, 133.2, 131.0,
129.9, 128.7, 128.5, 128.2, 127.6, 126.5, 126.1, 124.8, 122.9, 122.8, 122.5,
122.3, 117.2, 111.5, 49.1, 31.6, 29.1, 29.0, 28.9, 26.1, 22.5, 16.4, 14.34. HRMS
+
(ESI) calcd. for C31H33N2OS2 (M+) 513.2034, found 513.2031.
2.5 Theoretical calculations
In this investigation, merocyanine 8 was used as a model. In this sense,
the structures of the ground (S0) and first excited (S1) state were optimized
using Density Functional Theory (DFT) and Time-dependent DFT (TD-DFT),
respectively. For the optimizations, the functional B3LYP [40] including D3
Grimme’s dispersion corrections was used together with 6-31G(d,p) basis set
[41]. The equilibrium geometries were verified by vibrational analysis at the
same level of theory. At the equilibrium geometries, single-point calculations
were done at B3LYP-D3/6-311+G(d,p) level of theory. The vertical absorption
and emission energies were benchmarked against the experimental values
7

using several different DFT functionals. The better agreement between those
values was found at B3LYP-D3/6-311+G(d,p) level. The solvent effects were
included for the optimizations and vertical transitions by using the integral
equation formalism of the polarizable continuum model (IEF-PCM) and the
dielectric constants corresponding to 1,4-dioxane, dichloromethane, methanol,
and acetonitrile [42,43,44]. The ground and excited states molecular
electrostatic potential surfaces (MEP), charges, and dipole moment were
obtained by population analysis using the Charges from Electrostatic Potential
using a Grid-based method ChelpG [45]. The frontier molecular orbitals and
molecular structures were plotted using Chemcraft Software [46]. To evaluate
the intramolecular proton transfer, relaxed scans were done in the S1 state at
B3LYP/6-31G(d,p) level employing 1,4-dioxane and acetonitrile for the solvent
effect. For those, the geometries were optimized by increasing the O-H bond
distance by 0.05 Å each step. All DFT and TD-DFT calculations were performed
using Gaussian 16 Revision A.03 [47].
3. Results and Discussion
3.1 Synthesis
The merocyanine dyes 8-9 investigated in this work were synthesized by
condensation reaction between formylated benzothiazole 4 and quaternary
benzothiazole ammonium salts 6-7 as shown in Scheme 1. Preparation of 4
was performed through the previously described protocol in two steps [48].
First, the classical method of condensation between o-aminothiophenol (1) and
3-methylsalicylic acid (2), in equimolar amounts, in polyphosphoric acid (PPA)
leads to formation of benzothiazole 3 [49]. A second step of Duff formylation
reaction with hexamethylenetetramine (HMTA) in trifluoroacetic acid (TFA)
followed by the addition of water afforded formylated benzothiazole 4 with 84 %
yield. Noteworthy, formylation only occurs in 5’ position since ortho positions of
the phenolic ring are substituted with a methyl group and a benzothiazole
moiety [50].
The quaternary benzothiazole ammonium salts were obtained by a substitution
reaction between 2-methylbenzothiazole (5) and suitable alkylation agents. For
compounds 6 and 7 either methyl iodide (90% yield) or octyl iodide (44% yield)
8

respectively were used. Lastly, condensation of 4 with 6 and 7 yielded
merocyanine dyes 8 and 9 in 97 and 78%, respectively, after isolation by
precipitation. The reactions were performed in ethanol (efflux temperature) and
required one equivalent of base. Besides sodium methoxide, other bases as
sodium hydroxide and sodium carbonate were also tested and no product was
detected by TLC after 24 hours. During the reaction pathway, a color changing
of crude reaction from yellow to reddish is observed. It is highlighted the
1
presence of two doublets in H NMR spectra, for both merocyanines, attributed
to olefinic hydrogens with coupling constant (J) about 15.7 Hz that is in
agreement with the proposed trans conformer.
Scheme 1. Synthetic route for the preparation of compounds 8 and 9.
9

3.2 Photophysical study
The electronic properties of the compounds 8 and 9 were evaluated by
UV-Vis absorption and fluorescence emission spectroscopies in organic
solvents with different dielectric constants. The obtained photophysical data are
summarized in Table 2. In this investigation, the extinction coefficient ε was
correlated to the oscillator strength fe by the Strickler-Berg relations [51]. The
0
rate constant ke was also obtained using Equation 1, where the integral is
related to the area under the absorption curve corresponding to a single
electron oscillator. In addition, the pure radiative lifetime τ0 was also calculated,
defined as 1/ꢀꢁꢂ [52].
ꢀꢁꢂ ≈ 2.88ꢃ10ꢄꢅꢆꢇ
ꢋ ꢌ
̅ꢂ ꢈ ꢉꢊꢆ̅ , 1
Figure 1 presents the UV-Vis spectra from compounds 8 and 9 in
different organic solvents. It can be observed that changes on the substituent
(methyl or octyl) do not affect the absorption maxima wavelength due to the
similar electronic contributions of these lateral groups. The absorption maximum
is located between 585-605 nm with a medium negative solvatochromic effect
(∆λabs=30 nm). From the extinction coefficient and calculated radiative rate
constant values, the observed electronic transitions are fully spin and symmetry
1
allowed related to π-π* transitions. Moreover, the oscillator strength (fe) values
for 8 and 9 show almost unitary values, which corroborates with electronic
dipole-allowed transitions, as expected [53]. Practically constant emission
lifetime values (~10-9 s) indicate that after the absorption, the compounds
populate the same excited state.
10

Figure 2. UV-Vis absorption spectra of 8 and 9 in different solvents [~10-6M].
The normalized fluorescence emission spectra of compounds 8 and 9
are presented in Figure 2. The fluorescence emission data were obtained using
the absorption maxima as excitation wavelengths. The relevant data from this
investigation are also presented in Table 2. The studied compounds present
emission maxima located between 610-628 nm, once again, with a small
negative solvatochromic effect. Despite the presence of the 2-(2'-
hydroxyphenyl)benzothiazole core, able to proton-transfer in the excited state
(ESIPT) (Scheme 2), the studied merocyanines present a main and sharp
fluorescence emission with small Stokes shift (<1000 cm-1). This result is
contrary to expectations for ESIPT-containing compounds where a dual
fluorescence emission or even a main emission at longer wavelengths is
observed [54,55]. Surprisingly, this result indicates that the fluorescence
emission arises from a locally excited state, related to an enol conformer
(Scheme 2), discarding the possibility of proton transfer as effective deactivating
channel. This latter would lead to an emission with very large Stokes shift,
usually called as keto or ESIPT emission (Scheme 2).
Moreover, studies with very similar structures indicate that proton transfer
occurs in organic media and in PBS buffer [56-58], unlike what was observed in
this study. Thus, in this case, it is believed that the substitution pattern in the
phenolic ring tailor significantly the photophysical properties in these "virtually-
able-to-proton-transfer" merocyanines, indicating once again that the ESIPT
11

reaction is a particular and always interesting theme of investigation. It is worth
mentioning that excitation at higher energy (~400 nm) produced fluorescence
emission with the same maxima location, as expected for a compound that
follows the Kasha's rule. In this way, these additional blueshifted absorption
bands can be related to electronic transitions to excited states higher than S1
(see ESI).
Figure 3. Normalized fluorescence emission spectra of 8 and 9 in different
solvents [~10-6M] (Exc./Em. slits 5.0nm/5.0 nm).
Scheme 2. Förster cycle for the ESIPT process using compound 8 as a model.
The asterisk indicates the excited state and E and K indicate enol conformer
and the keto tautomer, respectively.
12

Table 2. General photophysical data of merocyanines 8 and 9, where ε is the molar extinction coefficient (x104 M-1·cm-1), λabs is the
0
absorption maxima (nm), fe is the calculated oscillator strength, ke is the calculated radiative rate constant (108 s-1), τ0 is the
calculated pure radiative lifetime (ns), λem is the emission maxima (nm), ∆λST is the Stokes shift (nm/cm-1) and QY is the
fluorescence quantum yield (%).
Merocyanine
Solvent
1,4-Dioxane
Dichloromethane
Ethanol
fe
k0
QY
0.6
1.9
1.5
1.2
0.8
1.4
1.6
1.7
ε
λabs
604
599
576
585
605
600
577
585
τ0
λem
628
622
611
610
626
621
611
610
∆λST
e
17.7
3.96
17.3
8.99
14.8
2.80
11.2
1.17
0.88
0.85
0.90
0.76
0.83
0.58
0.54
0.52
2.42
2.38
2.72
2.22
2.26
1.61
1.62
1.52
4.13
4.20
3.68
4.50
4.43
6.23
6.19
4.13
24/633
23/617
35/994
25/701
21/554
21/564
34/964
25/701
8
Acetonitrile
1,4-Dioxane
Dichloromethane
Ethanol
9
Acetonitrile
13

In order to investigate the presence of an intramolecular charge transfer
(ICT) mechanism in these compounds, the Lippert-Mataga correlation was
applied [59]. In this methodology, a solvent polarity plot function is obtained
[59,60], allowing the calculation of the difference in the dipole moment between
the excited and ground states (∆ꢍꢁꢇꢎ) (Equation 2). The linear plot of the
absorbance, fluorescence emission or Stokes shift versus the solvent polarity
function indicates the intramolecular charge transfer character [61].
ꢔ
ꢇ∆ꢑ
∆ꢆꢏꢐ
̅
=
ꢒꢓ ∆ꢙ + ∆ꢆꢂ
̅
(2)
ꢘ
ꢕꢖꢗ
In Equation 2, h is the Planck's constant, c is the speed of light and a is
the Onsager cavity radius. Aditionally, the orientation polarization function (∆f)
can be obtained from Equation 3 [62]:
ꢔ
ꢋ
ꢌ
ꢝ
ꢟ
ꢞ ꢄꢛ
ꢚꢄꢛ
∆ꢙ =
−
ꢌ , (3)
ꢔ
ꢋ
ꢌ
ꢋ
ꢇꢚꢜꢛ
ꢇꢞ ꢜꢛ
where the dielectric constant (ε) and the refractive index (n) for a mixture of
solvents can be calculated by Equations (4) and (5), where fA and fB are the
volumetric fractions of the two solvents.
ꢉꢠꢡꢢ = ꢙ ∙ ꢉꢣ + ꢙ ∙ ꢉꢤ (4)
ꢣ
ꢤ
ꢥꢠꢇ ꢡꢢ = ꢙ ꢥꢇ + ꢙ ꢥꢇ (5)
ꢣ
ꢤ
ꢤ
ꢣ
From the Lippert-Mataga correlation applied in the synthesized
compounds 8 and 9, there is no evidence for charge transfer in the studied
merocyanines in both ground and excited states (data not shown, see ESI),
which corroborates with the emission from the locally excited states. In addition,
it can be observed that these compounds are more destabilized in more polar
environments.
14

3.3 Ionization constant
The ionization constant (Ka) of these compounds was obtained by the
Henderson-Hasselbach relation (Equation 2) [32,63] from the acid-base
equilibrium in ethanol/water solutions (1:1 v/v).
ꢦꢧ = ꢦꢨꢗ + ꢩꢪꢫ ꢋꢣꢄꢣ ꢌꢌ , (2)
ꢬ
ꢋ
ꢣ ꢄꢣ
ꢭ
where Aa and Ab are the absorbances of the protonated and the neutral species
in basic media (KOH), respectively. In this equation, A is the absorbance from
the protonated and neutral species [64-66]. The determination of the respective
ionization constants by UV-Vis absorption spectroscopy takes the direct
determination of the ratio of the neutral to ionized species into account. In this
way, an absorption wavelength must be preferred where the difference between
the absorbances of the two species is maximized, as already observed in the
literature [20,67]. Thus, Figure 5a presents the UV-Vis absorption of
merocyanines 8-9 in ethanol/water solution (1:1 v/v) in two different pH values.
The neutral and protonated species show the maximum absorption wavelength
located at 418 and 420 nm, respectively. Based on these results, it is believed
that 560 (8) nm and 552 (9) nm are the most suitable wavelengths to calculate
the pKa for these compounds. Figure 5b shows the dependence of the
absorbance on the pH observed at 560 nm (8) and 552 nm (9), probably due to
the presence of the neutral and deprotonated species at different pH values.
The obtained results could be fitted using a sigmoidal curve, where the pH
values, 6.03 (8) and 6.18 (9), were obtained according to Equation (2) as the
inflection point (Table 3). These results indicate that the merocyanines 8-9 can
be explored for optical sensing in these pH values.
15

Figure 5. (a) UV-Vis absorption spectra of compounds 8 and 9 in ethanol/water
solution (1:1 v/v) in extreme pH values at 25oC. (b) The plot of the absorbance
intensity recorded at indicated wavelength versus pH. The dotted line was fitted
according to the Boltzmann Function - sigmoidal curve. The colorimetric
dependence of compounds 8 and 9 with the pH in 1:1 v/v ethanol/water solution
at high (~14) and low pH (~0.5) values by naked eye detection was also
presented for comparison..
Table 3. Photophysical data of 8 and 9, where Conc. is the concentration (10-6
M), λabs is the absorption maxima (nm), pKa is the ionization constant
ethanol/water solution (1:1 v/v) and wpKa is the ionization constant extrapolated
to water media.
λabs
Neutral form
418
λabs
Comp.
Conc.
pKa
wpKa
Deprotonated form
8
9
3.61
5.15
556
555
6.03
6.18
5.82
5.97
420
The ionization constant in water (wpKa) was determined from the pKa at the
same conditions (ethanol/water solution (1:1 v/v)) by applying Equation (3):
pKa = wpKa - δ, (3)
16

where δ is equal to 0.21 with 0.1 ionic strength by the addition of 0.15 mol·L-1
KCl [31,32,68-71]. Although non-substituted phenolic compounds present pKa
values around 9, as can be observed in Table 3, pKa ~6 were obtained, which
can be related to the presence of their substituents. Finally, their absorption in
the visible region, as well as the significative difference between the neutral and
deprotonated species observed on their absorption spectra, make these
compounds to present interesting and exploitable colorimetric response with the
pH. The protonated specie is yellow with absorption shifted to shorter
wavelengths while the deprotonated one is violet.
3.4 Theoretical calculations
Since changes in the lateral group R on the compounds 8 and 9 do not
affect the location of the experimental absorption maxima (see Table 2), as
already discussed, (TD)-DFT calculations were done only for the merocyanine
8. In this way, two different optimized conformers were found in the ground and
first excited states, which correspond to the sulphur atoms on the benzothiazole
ring in trans or cis position each other. Due to the presence of the hydroxyl
group attached to the central ring, enol and keto tautomeric structures were also
analyzed in the S1 state, as expected to ESIPT reactive compounds. The
optimized geometries in acetonitrile are shown in Figure 6. The cartesian
coordinates in acetonitrile, as well as the optimized structures in
dichloromethane, 1,4-dioxane, and ethanol and respective energies can be
found as supplementary data.
17

Figure 6. Optimized geometries (front and lateral view) in acetonitrile for the
ground and the first excited state of merocyanine 8.
In the S0 state, in all the solvent media investigated, the most stable
conformer presents the sulphur atoms oriented towards the same side
(Conformer 2, cis). This conformer is about 2.2 to 2.6 kcal·mol-1 more stable
than the trans conformer (Conformer 1). This could be attributed to the more
extended conjugation in Conformer 2. In the last one, the hemicyanine moiety is
coplanar and conjugated with the phenyl moiety and HBT core through an
ethylene bridge, while in Conformer 1 the cyanine moiety is about 30 degrees
out-of-plane. As a consequence of the increased conjugation, the ethylene
moiety in Conformer 2 has smaller bond length alternation (BLA) [72] than
conformer 1 due to the most effective delocalization of π-electrons. The same
effect is observed in the S1 state (for the enol and keto tautomers), where the
planar Conformer 2 is even more stable (by 3.3 to 3.5 kcal·mol-1) when
compared to Conformer 1. Going from the S0 to the S1 state (enol conformer)
18

the main geometric changes observed are the smaller BLA in the ethylene
bridge, as well as changes in the bond lengths in the central part of the
molecule comprised between the two thiazole rings (bond lengths are more
similar than in the S0 state). Interestingly, enol tautomer was found to be
preferred in 1,4-dioxane by ~2.3 kcal·mol-1, while in the remaining solvents the
keto tautomer was favored by 2.3 to 2.7 kcal·mol-1.
The frontier molecular orbitals (FMOs) plotted in acetonitrile are shown in
Figure 7. The data in 1,4-dioxane is presented as supplementary material. It
can be observed that in the S0 state, the HOMO is delocalized over the whole
molecule, while the LUMO is mainly delocalized over the merocyanine and the
vinyl bridge. The vertical electronic transitions have ππ* character and large
oscillator strengths indicating that the S1 state is the bright state. Interestingly, in
the S1 state, the HOMO associated with the calculation in 1,4-dioxane is slightly
different than in the other solvents, being more localized over the HBT unit while
in the other solvents, HOMO is delocalized over the whole molecule. This result
shows that, in 1,4-dioxane, the S1 state presents a smaller locally excited (LE)
character and it should have some ICT character. Besides polymethine dyes
are known to have small Stokes shift (<25 nm) when they do not present
excited-state ICT between the donor and acceptor units [73], results indicating
ICT were presented by Li et al. for donor-two-acceptor hemicyanine-HBT based
fluorophore [74]. Molecule 8 can be classified as one-donor-two-acceptors
systems because of the presence of the hydroxyl group. Yet, due to the fact that
the electron-donating hydroxyl group can be involved in both ICT and ESIPT
processes, deprotonation can modulate both processes.
19

Figure 7. Frontier Molecular Orbitals (HOMO/LUMO) calculated for both ground
and excited (Enol) states in acetonitrile for Conformer 2 of merocyanine 8.
Despite the presence of 2-(2’-hydroxyphenyl)benzothiazole group, and
the large literature about experimental evidence on proton transfer in this
fluorophore in different structures [75-80] or media [
Error! Bookmark not
,81-86], ESIPT was not observed in this compound. This can be
defined.
explained by the larger barriers for the proton transfer (Figure 8). In fact, the
calculations in 1,4-dioxane show again a different pattern, where the enol is
more stable than keto tautomer, in contrast to the calculations in the other
solvents. However, in all the cases, the presence of a barrier larger than 2.0
kcal·mol-1 suggests that the enol tautomer should be the most probable one. In
addition, looking at the charge distribution on the nitrogen and oxygen atoms
potentially involved in the ESIPT (see Table S3), it can be seen that the
negative charge on the oxygen atom slightly decreases in the S1 state while the
charge in the nitrogen is less affected. The small change in the charge
20

distribution in N1 and O1 also indicates that excited state intramolecular proton
transfer in this molecule is not probable to happens.
Figure 8. Potential energy surface in 1,4-dioxane and acetonitrile for the
intramolecular proton transfer in the S1 state.
The molecular electrostatic potential (MEP) surfaces of compound 8 in
acetonitrile show that there is no substantial separation in the overall charge
distribution in such media (Figure 9). Similar behavior was observed in 1,4-
dioxane (data not shown, see Figure S29). This result agrees with the very
small solvatochromic effect experimentally observed. When comparing the MEP
surfaces in the S0 and S1 states, it is clear that the charge separation is higher
in the S0 than in the S1 state. This is reflected on the larger dipole moment in
the ground state (µdioxane= 13.0; µacetonitrile = 15.9) than in the excited state
(µdioxane= 7.6; µacetonitrile = 7.4) and in the consequent small negative
solvatochromic effect observed (see Table S2). In this case, solvents with
higher polarity stabilize the S0 better than the S1 state, which increases the
energy gap and, therefore, increases the maximum wavelength.
21

Figure 9. Molecular electrostatic potential (MEP) surfaces of 8 (conformer 2) in
the ground (left) and excited (right) states calculated in acetonitrile.
4. Conclusions
The novel merocyanine dyes containing two benzothiazole conjugated units
were easy synthesized by robust condensation protocols. The photoactive
compounds were obtained in excellent yields and present and simple
purification. The compounds present electronic transitions in the green-orange
region ascribed to fully spin and symmetry allowed and fluorescence emission
at the orange red region with very small Stokes shift. No evidence for ESIPT as
well as for intramolecular charge transfer in the excited state was observed.
Theoretical calculations show that the S1 state has a local excited character and
ESIPT is not probable to happen in these compounds due to the high energy
barriers for transferring the proton. By changing the pH value, the studied
compounds present significant changes in the absorption spectra (yellow to
violet color), which allow their application as optical sensors for middle pH
values
Acknowledgments
The authors would like to acknowledge FAPERGS (17/2551-0000968-1), CNPq
(409855/2018-9) and Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - Brasil (CAPES) - Finance Code 001 for the financial support.
22

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