Bimolecular fluorescence quenching of benzoxazole/benzothiazole-based functional dyes

Article abstract of DOI:10.1016/j.molliq.2020.113489

Four dyes that belong to the group of hemicyanines and possess benzoxazole and benzothiazole moieties are synthesised and described. The spectroscopic properties of these compounds in 1-methyl-2-pyrrolidinone (MP) were studied. Synthesised dyes absorb in the UV–Vis region. The emission spectra are broad with a maximum located at approximately 500–520 nm. The deactivation of excited states of the synthesised molecules by various quenchers is also presented. The fluorescence quenching parameters were calculated using the Stern-Volmer equation. It was found, that the fluorescence quenching of the excited dye by quencher molecules occurs. The fluorescence quenching rates (k_q) are about 10¹⁰ M^?1 × s^?1.

Full text of DOI:10.1016/j.molliq.2020.113489

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Bimolecular
fluorescence
quenching
of
benzoxazole/
benzothiazole-based functional dyes
Alicja Balcerak, Janina Kabatc
PII:
S0167-7322(20)31395-7
DOI:
https://doi.org/10.1016/j.molliq.2020.113489
MOLLIQ 113489
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
6 March 2020
22 May 2020
30 May 2020
Please cite this article as: A. Balcerak and J. Kabatc, Bimolecular fluorescence quenching
of benzoxazole/benzothiazole-based functional dyes, Journal of Molecular Liquids
(2020), https://doi.org/10.1016/j.molliq.2020.113489
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Bimolecular fluorescence quenching of benzoxazole/benzothiazole-based functional dyes
Alicja Balcerak, Janina Kabatc*
UTP University of Science and Technology, Faculty of Chemical Technology and Engineering,
Seminaryjna 3, 85-326 Bydgoszcz, Poland
ABSTRACT
Four dyes that belong to the group of hemicyanines and possess benzoxazole and
benzothiazole moieties are synthesized and described. The spectroscopic properties of these
compounds in 1-methyl-2-pyrrolidinone (MP) were studied. Synthesized dyes absorb in the UV-Vis
region. The emission spectra are broad with a maximum located at approximately 500-520 nm. The
deactivation of excited states of the synthesised molecules by various quenchers is also presented.
The fluorescence quenching parameters were calculated using the Stern-Volmer equation. It was
found, that the fluorescence quenching of the excited dye by quencher molecules occurs. The
fluorescence quenching rates (kq) are ~1010 M-1 × s-1.
Keywords: hemicyanine dyes, spectroscopic properties, fluorescence quenching, quencher, Stern-
Volmer equation
1. Introduction
Mauveine was the first discovered dye. In 1856 Perkin synthesised this compound and
started a new era of synthesis of dyes [1]. Since then, searching and designing new dyes using novel
methods of synthesis, which might exhibit unique and useful physico-chemical properties, became
very important for a wide group of scientists [2]. The dye industry is still developing, this is
indicated by the number of dyes produced. It is estimated, that more than 7×105 tons of synthetic
dyes are produced in the world with about 104 types of commercially available dyes [3, 4].
Cyanines (polymethines) have been of great interest for many years because of their
multidimensional applications. Typical cyanine dye is composed of a methine chain (-CH=CH-)n
with two aza-heterocyclic moieties, such as benzothiazoles, benzoxazoles, pyridines, quinolines,
indoles etc., which are attached to the conjugated double bond system. The introduction of
appropriate substituents instead of heterocycles results in diversification of structure and properties
of these compounds.
* Corresponding author: nina@utp.edu.pl (Janina Kabatc); Tel.: +48 52 374 9112; Fax: +48 52 374 9009
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Both, the type of terminal groups and the length of the polymethine chain affect the position of the
maximum absorption for cyanine dyes. This is related to delocalisation of electrons in the molecule,
which causes the absorption over a wide spectrum range, from visible to the near infrared region [5-
7].
Cyanines have narrow absorption bands and are characterised by very high molar extinction
coefficients. Moreover, these compounds display large microscopic polarisabilities, intensive
fluorescence in the visible region, high affinity to cell structures and the ability to form highly
ordered self-assemblies (J-aggregates) [8-12]. These unique properties aforementioned, allow their
use in other different aspects such as antitumor agents in Photodynamic Therapy (PDT) [13-15],
fluorescence probes in biological imaging [16, 17], sensitisers in dye-sensitised solar cells (DSSCs)
[18-20], sensors for detection of various substances [21-23], optical recording and data storage
media [24, 25], and many others.
Chemical molecules exhibiting intensive fluorescence are of interest in organic synthesis [26].
The study of the fluorescence phenomenon of molecules is extremely significant and shows huge
potential in the diagnosis and treatment of different types of cancer. This fact is confirmed by
numerous of articles presenting the justified application of dyes in PDT, which is defined as a
minimally invasive therapeutic technique.
For example, in 2020, Lima and co-workers [27] described novel indolenine-based squaraine
dyes that provide a potential application as photosensitisers in therapeutic systems against
carcinoma cells. These compounds display strong, sharp absorption within the so-called
“phototherapeutic window” (650-850 nm), high photostability and the ability for the generation of
singlet oxygen (1O2). The reactive oxygen species are the main cytotoxic agent, which induce
infected cell death via oxidative stress mechanisms, thus the system is selective [28].
In 2019 Bokan et al., presented a new method for targeted drug delivery (TDD) by means of a
combination an anticancer medicine with a proper fluorogenic dye. The novel, water-soluble
phenolo-cyanine dyes were bound to the chlorambucil (CLB) medicament through a biodegradable
ester linker. The developed conjugates possess a single reactive carboxylic group that is able to bind
with a specific target cancer carrier, e.g.antibodies or peptides. The hydrolytic cleavage of the ester
bond in the cell medium, causes the drug to be released and this fluorescent system exhibits
emission in over a wide region of the spectrum with a maximum located at 720 nm. Therefore, the
proposed molecules are really promising for tumour drugs delivery to target infected structures [29].
Because of the significant interest in looking for alternative renewable sources of energy, in
the last years, the development of dye-sensitised solar cells has been rapidly increased. In DSSCs,
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the sensitiser plays a key role in absorbing the light and initiating the electrochemical reactions for
electricity production [30].
Confirmation of the usability of cyanines as sensitisers in solar cells was explored by Ghann
and co-workers [18]. The group determined the photovoltaic efficiency of conversion to be 0.04%-
0.24%. In 2019, new systems containing organic dyes with quantum dots (QDs) were proposed. The
surface deposition of cyanine using QDs increased the efficiency of DSSCs to 0.89% in comparison
to typical solar cells only based on the dye [31].
The examples mentioned, confirm the important role of searching for new dyes and studying
their properties. These works show an application of novel designed dyes in various fields, such as
science, engineering, technology, pharmacy, and medicine. Due to a wide range of applications for
cyanine-based dyes, it is necessary to examine their features. In our study, we present spectral
properties and fluorescence quenching of four synthesised hemicyanines with different molecules
used as quenchers.
2. Experimental
2.1. Materials and general methods
All reagents for the synthesis of the hemicyanines and spectroscopic grade solvent: 1-
methyl-2-pyrrolidinone (MP) were purchased from Sigma-Aldrich (Poland) and used without
further purification. The commercially available compounds used as quenchers were purchased
from Sigma-Aldrich (Poland) or Alfa Aesar and the others were synthesised in our laboratory by
methods described in the literature [32, 33].
Absorption and emission spectra were recorded using an Agilent Technology UV-Vis Cary
60 Spectrophotometer and a Hitachi F-7000 Spectrofluorimeter, respectively. The measurements
were carried out at room temperature with 1-methyl-2-pyrrolidinone as the solvent.
The fluorescence quantum yield for each dye (Φdye) was determined relative to Coumarine I,
as a reference standard, and calculated based on the following equation [34]:
(1)
where: Φref is the fluorescence quantum yield of the reference, Idye and Iref are the integration of the
fluorescence intensity for the dye and reference sample, Adye, Aref are the absorbances of dye and
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reference at the excitation wavelength, ndye, nref refer to the refractive indexes of solvents used for
dye and reference, respectively.
The fluorescence lifetimes, as well as fluorescence quenching, were measured by the use of
a single-photon counting system UV-VIS-NIR Fluorolog 3 Spectrofluorimeter (Horiba Jobin
Yvon). This apparatus utilises molecule excitation with a picosecond diode laser (370 nm), which
generates pulses of ca. 55 ps. A successful analysis of fluorescence decay signals with a resolution
of few picoseconds is possible as a result of the short laser pulses in conjunction with fast
microchannel plate photodetector and ultrafast electronics. The concentration of the dye was fitted
to provide equivalent absorbance at excitation wavelength equal to 370 nm (0.2 in the 10 nm cell).
The rate constant for the quenching process of the hemicyanines by all quenchers were studied in 1-
methyl-2-pyrrolidinone. The concentration of quenchers varied from 1.0 × 10-4 M to 9.0 × 10-4 M
(for R1/I1 system the concentration of quencher was 10-times higher).
3. Results and discussion
3.1. Synthesis
Four hemicyanines were synthesised according to the well-known procedure based on
Knoevenagel condensation of a N-alkyl heterocyclic base containing active methyl group in the 2-
position with an aromatic aldehyde [33, 35]. The synthetic routes of compounds under study are
shown in Scheme 1. In general, the neutral dyes R1 and R2 were obtained by heating equimolar
amounts of 2-methylbenzoxazole or 2-methylbenzothiazole (respectively for R1 and R2) and p-
(N,N-dimethylamino)benzaldehyde in presence of boric acid (0.05 g per 0.01 M of aldehyde) at
0
195-200 C for 5h. The ionic dyes were synthesised by the heating of appropriate amounts of
0
compounds R1 or R2 with 1,3-butanesultone at 120 C for 1h. The products of the reactions were
crystallised from ethanol (neutral dyes) or mixture ethanol:hexane (10:1) and dried at ambient
temperature.
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X
N
O
H
CH3
X
N
CH3
CH3
boric acid
+
CH3
C
N
CH CH
N
CH3
Neutral dyes: R1, R2
where: X = O, S
O
O
O
S
X
N
CH3
CH3
CH CH
N
SO3
Ionic dyes: R3, R4
Scheme 1. General synthesis pathway of tested compounds R1-R4.
1
The structures of synthesised dyes were confirmed using H and 13C NMR spectroscopy (data
presented in [33]). These compounds belong to the group of hemicyanines (styryl dyes) possessing
two nitrogen atoms linked with a chain of conjugated double bonds, wherein one of nitrogen atom
is not a part of the heterocyclic ring [7]. In Fig. 1, basic structural information about the systems is
presented.
Hemicyanines
O
N
S
CH3
CH3
CH3
CH3
CH CH
N
CH CH
N
N
2-[p-(N,N-dimethylaminostyryl)]benzoxazole
2-[p-(N,N-dimethylaminostyryl)]benzothiazole
R1
R2
O
S
CH3
CH3
CH CH
N
CH CH
N
CH3
CH3
N
N
SO3
SO3
2-[p-(N,N-dimethylamino)styryl]benzoxazole
2-[p-(N,N-dimethylamino)styryl]benzothiazole
butanesulfonate
butanesulfonate
R3
R4
Fig. 1. Chemical structures, names and abbreviations of hemicyanine dyes.
3.2. Spectroscopic properties of hemicyanines
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The photophysical data of the hemicyanine dyes were studied and are summarised in Table
1. As can be seen in Fig. 2, the compounds absorb in the UV-Vis region and emit between 450 nm
to 600 nm range. The visible band of absorption arise from electronic transitions involving π
electrons along the polymethine chain. Generally, the dyes exhibit a maximum absorption (λab)
band at about 400 nm. For R4, it was observed that the second maximum of absorption is located at
530 nm. The emission spectra are single and broad with a maximum of fluorescence (λfl) located at
about 500 nm for benzoxazole-based dyes and at ca. 520 nm for the benzothiazole derivatives.
Moreover, the Stokes shifts achieved relatively high values. These characteristics suggest that for
fluorescence lifetimes, the crucial structural changes take place in the S1 state of the solvated
molecule relative to the primary state S0.
R1
R2
R3
R4
R1
R2
R3
R4
1000
800
600
400
200
0
1,0
0,8
0,6
0,4
0,2
0,0
350
400
450
500
550
600
400
450
500
550
600
650
700
Wavelength [nm]
Wavelength [nm]
Fig. 2. Normalised absorption and fluorescence spectra of the tested dyes recorded on 1-methyl-2-pyrrolidinone
(MP) at room temperature.
High molar extinction coefficients for the synthesised benzoxazole and benzothiazole
derivatives were determined. The fluorescence quantum efficiency (Φfl) was calculated from the
ratio of the number of photons emitted by the molecule to the number of absorbed [36]. The
quantum yields achieved quite low values and the excited state energy levels (E00) are similar for all
tested dyes. Low values of Φfl may be indicative of the radiative or non-radiative deactivation
processes that transform the molecule from the excited to ground state.
Table 1
Spectroscopic properties of studied dyes in 1-methyl-2-pyrrolidinone (MP).
Parameter
Dye
λab
FWHMa
b [cm-1]
ε
λfl
FWHMfl
[cm-1]
Stokes shift
[cm-1]
Φfl
[10-2]
E00
[nm]
[104 M-1 × cm-1]
[nm]
[eV]
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R1
R2
R3
R4
399
406
4642
4257
4303
4006
3.61
498
521
496
517
3116
4982
5437
4901
5167
1.59
2.03
1.92
1.54
2.77
2.65
2.78
2.64
2.30
4.99
3.08
3106
3517
3290
399
408 (530)
3.3. Deactivation of excited states of cyanine dyes
Absorption of light results in excitation of molecule and transition from ground to excited
singlet state. Methods for deactivation of excited states depend on the type of absorbing molecule.
As presented in Scheme 2, there are major quenching routes from the excited singlet to ground
singlet state for typical polymethines:
 single–molecule deactivation: fluorescence, internal conversion, and intersystem crossing.
 bimolecular deactivation: photoisomerisation and reaction between the excited state of dye and
quencher molecule [37].
Photoisomerization
X
CH3
CH3
*
CH CH
N
N
S2
S1
Y
X
CH3
CH3
*
Internal conversion
(IC)
CH CH
N
N
Y
Excited state of dye
molecule
Quencher
Intersystem
crossing (ISC)
T1
Energy
Light
Internal
conversion
(IC)
Absorption
Fluorescence
X
CH3
CH CH
N
CH3
N
Y
Singlet state of dye
molecule
S0
Bimolecular deactivation
Single-molecule deactivation
Scheme 2. Simplified energy level diagram of dyes under study showing routes for deactivation of an
excited state of the molecule.
Fluorescence quenching (FQ) is defined as the physicochemical process of relaxation, that
occurs without photon emission. This process results in the reduction of the fluorescence lifetime
and fluorophore intensity. Deactivation of the excited state of the molecule can be directly
monitored by measurement of fluorescence lifetime as a function of intensity vs time the after light
excitation [38-40].
FQ of fluorophores, using different molecules acting as the quenchers, has been widely
studied to understand the mechanism of reaction that occurs in transient and steady-state conditions
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[41]. The excited state quenching is usually classified into three major groups: static, dynamic, and
resonance energy transfer. Generally, the quenching of the fluorescence process can be studied
experimentally by estimating the characterizing rate parameters on the basis of the Stern-Volmer
equations, which are presented below [42, 43]:
(2)
[ ]
where: I0 and I are the fluorescence intensities of solute molecules in the absence and in the
presence of a quencher, respectively, KSV is the Stern-Volmer (S-V) constant, and [Q] is the
concentration of quencher.
The KSV constant is expressed by Eq. (3):
(3)
where: kq is the quenching rate parameter, and τ0 is the lifetime of the fluorophore in the absence of
quencher.
Equation (2) is appropriate for linear variation of the obtained experimental results.
Fluorescence quenching of excited state of dye – fluorescence lifetime
As summarised in Table 2, the synthesised hemicyanines show similar fluorescence
lifetimes. The emission lifetime of the molecule is short and occurs within ca. 6 ns. This is an
impermanent phenomenon, which is typical for organic molecules.
Table 2
Fluorescence lifetime values determined for R1-R4 dyes.
Dye
R1
R2
R3
R4
Fluorescence lifetime [10-9 s]
6.75
5.66
5.82
6.58
The FQ in the presence of the quencher is more effective than that observed for dye without a
quencher due to the possibility of a strong interaction between the singlet fluorescent state of
fluorophore and quencher.
Bimolecular fluorescence quenching
In order to explain the effect of other molecules on the photochemical behaviour of the light-
absorbing dyes, experiments based on fluorescence quenching of dyes as a function of quencher
concentration were carried out. The compounds used as fluorescence quenchers are presented in
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Fig. 3. All quenchers used in the experiments are not fluorescent compounds compared to the R1-
R4 dyes that undergo a radiation deactivation after excitation. These compounds belong to
diphenyliodonium salts, N-alkoxypyridinium salts, borate salt, amines and 1,3,5-triazine and were
also usually used as co-initiators in photoinitiating systems for radical polymerisation of various
acrylate monomers [44, 45].
Quenchers
F3C
I
OCH3
H3CO
I
NO2
CH3
N
OCH3
I
CH3
BF4
Cl
SO3
SO3
diphenyliodonium chloride
(4-methoxyphenyl)-(4-
nitrophenyl)iodonium p-
toluenesulfonate
(4-trifluoromethylphenyl)-
N-methoxy-4-
phenylpyridinium
tetrafluoroborate
(4-
methoxyphenyl)iodonium
p-toluenesulfonate
I85
I1
I81
NO
S
N
OCH3
N
B
SH
N
N(CH3)4
BF4
CH
CH2
N-methoxyisoquinoline
tetramethylammonium n-
N-vinylcarbazole
2-mercaptobenzothiazole
tetrafluoroborate
butyltriphenylborate
NO2
B2
NVK
MS
Cl
F
F
Cl
F
O
N
O
H
F
CH3
N
O
CH3
N
N
F
N
F
OH
H3C
HO
OH
N
CH3
Cl
2,4,6-
N-methyl-N,N-
ethyl (N,N-
N-phenylglycine
tris(chlorodifluoromethyl)-
1,3,5-triazine
diethanolamine
dimethylamino)benzoate
T1
MDEA
EDMAB
NPG
Fig. 3. Chemical structures, names, and abbreviations of the quenchers used in the study.
Figure presents the fluorescence decay profiles of 2-[p-(N,N-
4
dimethylaminostyryl)]benzoxazole (R1) in the presence of different concentrations of N-
methoxyisoquinoline tetrafluoroborate (NO2), used as a quencher. The quencher has a significant
effect on the fluorescence lifetime. As a result of the interaction between the dye and quencher, a
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shortening of the fluorescence lifetime was observed. An increase in the concentration of the
quencher decreases the fluorescence lifetime of the dye.
7000
1 x 10-4
3 x 10-4
5 x 10-4
7 x 10-4
9 x 10-4
M
M
M
M
M
6000
5000
4000
3000
2000
1000
0
O
N
CH3
CH3
CH CH
N
N
OCH3
BF4
16
18
20
22
24
26
28
30
Time [ns]
Fig. 4. Fluorescence decay profile of R1 in the presence of various concentrations of NO2.
The data obtained from FQ experiments are discussed on the basis of the Stern-Volmer
relationship. The characterizing parameters from the S-V equation are summarised in Table 3.
Table 3
The values of the Stern-Volmer constant (KSV) and rate constant of fluorescence quenching (kq) for the
systems in this study.
Quencher
Dye Parameter
I1
I81
I85
NO
NO2 B2
NVK MS
T1
MDEA NPG EDMA
KSV [102 M-1] 0.47 3.12 6.18 4.73 11.21 9.14 3.69 5.69 4.90 7.39 7.32 6.85
kq [1010 M-1 × s 0.70 4.62 9.15 7.00 16.60 13.54 5.46 8.42 7.25 10.94 10.84 10.15
KSV [102 M-1] 2.73 2.88 3.09 3.67 6.43 5.61 2.06 3.81 3.27 1.57 3.42 3.50
kq [1010 M-1 × s 4.82 5.09 5.45 6.48 11.35 9.90 3.63 6.72 5.77 2.78 6.05 6.17
R1
R2
R3 KSV [102 M-1] 4.46 4.46 5.09 4.56 3.14 7.54 3.76 5.03 4.25 5.47 5.72 5.65
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kq [1010 M-1 × s 7.66 7.65 8.74 7.84 5.39 12.95 6.47 8.63 7.31 9.40 9.83 9.71
KSV [102 M-1] 4.03 5.46 5.95 3.91 6.34 6.82 3.54 6.66 4.77 6.25 8.77 5.92
kq [1010 M-1 × s 6.12 8.29 9.04 5.94 9.63 10.37 5.37 10.12 7.25 9.50 13.33 9.00
R4
From the data presented in Table 3 and Figure 5, it is seen, that the rate of fluorescence
quenching depends on the type of quencher used. It can be concluded, that there are strong
interactions with the dye-quencher. A linear relationship between the change in the fluorescence
lifetime and concentration of the quencher molecule is observed. The gradients of the Stern-Volmer
relationship achieve relatively high values. This parameter is over a range from 0.47 × 102 M-1 to
11.21 ×102 M-1. The most efficient quenchers for the neutral dyes are tetramethylammonium n-
butyltriphenylborate (B2), N-methoxyisoquinoline tetrafluoroborate (NO2), and borate salt (B2) or
N-phenylglycine (NPG) for ionic dyes R3 and R4, respectively. The highest values of the Stern-
Volmer constant were observed when NO2 and B2 were used as quenchers for the excited state of
neutral dyes R1 and R2. In the case of dyes R3 and R4, the highest values of KSV were recorded for
the borate salt (B2) and N-phenylglycine.
2,0
I1
I81
I85
1,8
1,6
1,4
1,2
1,0
NO
NO2
B2
NVK
MS
T1
MDEA
NPG
EDMAB
0,0000
0,0002
0,0004
0,0006
0,0008
0,0010
Quencher concentration [M]
Fig. 5. The Stern-Volmer plots for the quenching of the fluorescence for 2-[p-(N,N-
dimethylamino)styryl]benzothiazole butanesulfonate (R4) by all the quenchers outlined.
Based on Eq. 3, fluorescence lifetimes, and the Stern-Volmer constants the fluorescence
quenching rate constants (kq) were calculated (see Table 3 and Table 2). It was found, that this
parameter is in the range from 0.70 × 1010 M-1 × s-1 for iodonium salt (I1) to 16.60 × 1010 M-1 × s-1
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for tetrafluoroborate (NO2). The correlation coefficients (R2) in all cases for the dye/quencher
systems range from 0.98 to 0.99. Generally, with increasing concentration of quencher, the kq
parameter increases. It can be concluded that all used molecules are fast and effective quenchers for
the excited state of the dye. These promised values of S-V parameters may be due to the formation
of an intramolecular pair which results in quenching of the fluorescence process. The quenching
mechanism is based on an electron transfer process between the system's components.
4. Conclusions
In this paper, the fluorescence quenching process of hemicyanines by various quenchers is
presented. All used molecules possess the ability for quenching of excited states in R1-R4 dyes. The
Stern-Volmer plots confirm strong interactions between the dye and quencher that leads to high
values of the fluorescence quenching rate constant parameter. The kinetics of bimolecular
fluorescence quenching depends on the type of quencher. The linear S-V correlation shows that the
efficiency of quenching is associated with the concentration of quencher.
Conflict of interest
The authors have no conflicts of interest to declare.
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Author Statement
Declaration of Interest: The authors certify that they have no commercial or associative interest that
represents a conflict of interest in connection with the manuscript.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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Graphical abstract
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Highlights


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
Spectroscopic characteristic of benzoxazole and benzothiazole-based dyes .
Bimolecular fluorescence quenching of hemicyanines.
Kinetic parameters of radiation deactivation depend on the type of quencher.
The Stern-Volmer relationship between fluorescence lifetime and concentration of quencher.
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