Non-typical fluorescence effects and biological activity in selected 1,3,4-thiadiazole derivatives: Spectroscopic and theoretical studies on substituent, molecular aggregation, and pH effects

Article abstract of DOI:10.3390/ijms20215494

The below article presents the results of spectroscopic research, theoretical (timedependent density functional theory (TD-DFT)), microbiological, and antioxidative calculations for three compounds from the group of 1,3,4-thiadiazoles: 2-amino-5-phenyl-1,

Full text of DOI:10.3390/ijms20215494

International Journal of
Molecular Sciences
Article
Non-Typical Fluorescence Effects and Biological
Activity in Selected 1,3,4-thiadiazole Derivatives:
Spectroscopic and Theoretical Studies on Substituent,
Molecular Aggregation, and pH Effects
Iwona Budziak 1, Dariusz Karcz 2 , Marcin Makowski 3, Kamila Rachwał 4, Karolina Starzak 2
Alicja Matwijczuk 5 , Beata Mys´liwa-Kurdziel 6 , Anna Oniszczuk 7, Maciej Combrzyn´ski 8
,
,
Anna Podles´na 9 and Arkadiusz Matwijczuk 5,
*
1
Department of Chemistry, University of Life Sciences in Lublin, 20-950 Lublin, Poland;
iwona.budziak@up.lublin.pl
Department of Analytical Chemistry (C1), Faculty of Chemical Engineering and Technology, Cracow
University of Technology, Warszawska 24, 31-155 Cracow, Poland; dkarcz@chemia.pk.edu.pl (D.K.);
kstarzak@chemia.pk.edu.pl (K.S.)
2
3
4
5
6
7
8
9
Department of Theoretical Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2,
30-387 Krakow, Poland; makowskm@chemia.uj.edu.pl
Department of Biotechnology, Microbiology and Human Nutrition, University of Life Sciences in Lublin,
Skromna 8, 20-704 Lublin, Poland; kamila.rachwal@up.lublin.pl
Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland;
alicja.matwijczuk@up.lublin.pl
Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, 30-387 Krakow, Poland; b.mysliwa-kurdziel@uj.edu.pl
Department of Inorganic Chemistry, Medical University in Lublin, 20-059 Lublin, Poland;
anna.oniszczuk@umlub.pl
Department of Thermal Technology and Food Process Engineering, University of Life Sciences in Lublin,
20-950 Lublin, Poland; maciej.combrzynski@up.lublin.pl
Department of Plant Nutrition and Fertilization, Institute of Soil Science and Plant Cultivation-State
Research Institute, 24-100 Pulawy, Poland; ap@iung.pulawy.pl
*
Correspondence: arkadiusz.matwijczuk@up.lublin.pl; Tel.: +(48-81)-445-69-37; Fax: +(48-81)-4456684
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 September 2019; Accepted: 30 October 2019; Published: 4 November 2019
Abstract: The below article presents the results of spectroscopic research, theoretical (time-dependent
density functional theory (TD-DFT)), microbiological, and antioxidative calculations for three
compounds from the group of 1,3,4-thiadiazoles: 2-amino-5-phenyl-1,3,4-thiadiazole (TB), 2-amino-5-
(2-hydroxyphenyl)-1,3,4-thiadiazole (TS), 2-amino-5-(2-hydroxy-5-sulfobenzoyl)-1,3,4-thiadiazole
(TSF). In the fluorescence emission spectra (TS) of solutions with varying concentrations of hydrogen
ions, a particularly interesting effect of dual fluorescence was observed. The aforementioned effect was
observed even more clearly in the environment of butan-1-ol, relative to the compound’s concentration.
Depending on the modification of the resorcylic substituent (TS and TSF), we observed the emergence
of two separate, partially overlapping, fluorescence emission spectra or a single emission spectrum.
Interpretation of the obtained spectra using stationary and time-resolved spectroscopy allowed the
correlation of the effect’s emergence with the phenomenon of molecular aggregation (of a particular
type) as well as, above all, the structure of the substituent system. The overlap of said effects most
likely induces the processes related to the phenomenon of charge transfer (in TS) and is responsible
for the observed fluorescence effects. Also, the position of the –OH group (in the resorcylic ring)
is significant and can facilitate the charge transfer (CT). The determinations of the changes in the
dipole moment and TD-DFT calculations further corroborate the above assumption. The following
paper presents the analysis (the first for this particular group of analogues) of the fluorescence
effects relative to the changes in the structure of the resorcylic group combined with pH effects.
Int. J. Mol. Sci. 2019, 20, 5494; doi:10.3390/ijms20215494
www.mdpi.com/journal/ijms

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The results of biological studies also indicate the highest pharmacological potential of the analogue
in the case where the effects of dual fluorescence emission are observed, which predisposes this
particular group of fluorophores as effective fluorescence probes or potential pharmaceuticals with
antimycotic properties.
Keywords: molecular aggregation; 1,3,4-thiadiazole; molecular spectroscopy; dual fluorescence
effects; effect of the substituent group structure
1. Introduction
Contemporary medicine related to the treatment of conditions such as neoplastic,
neurodegenerative, or mycotic diseases requires the application of scientific methods relying on
the techniques of molecular spectroscopy and photophysics/photochemistry to provide an in-depth
description of the physicochemical properties of new compounds offering the aforementioned
properties. Therefore, both in the context of medicine and pharmacology, researchers employing the
methods of molecular spectroscopy are faced with the very difficult and highly engaging task (also
requiring specific specialist knowledge) of identifying new compounds displaying very particular
molecular properties, oftentimes providing a multi-directional range of specific pharmacological effects.
The selected molecules need to be very thoroughly analyzed in terms of their photophysical and
photochemical properties to allow the identification of the analogues with the highest potential for
actual medical applications.
Particular promise in the fight against the aforementioned diseases is shown by the compounds
from the group of 1,3,4-thiadiazoles substituted with a resorcylic group (Scheme 1A–F).
A
C
E
B
D
F
Scheme 1. Chemical structure of compounds (
A
,B
) benzoic thiadiazol (TB); (
C
,D) salicylic thiadiazol
(TS); (E,F) sulfosalicylic thiadiazol (TSF) and their interaction between molecules (dimers).
1,3,4-Thiadiazoles are a group of new potential pharmaceuticals showing neuroprotective [1],
anti-cancer [2], antibacterial [3], antioxidative [4], and antimycotic [3] properties. The following
paper presents the results of spectroscopic analyses as well as theoretical and biological calculations
conducted for three analogues from the 1,3,4-thiadiazole group.
For the performed study on the mechanisms of molecular interactions in aqueous solutions of
varying pH as well as in selected organic solvents (of varying concentration), the following compounds

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were selected: 2-amino-5-phenyl-1,3,4-thiadiazole (TB), 2-amino-5-(2-hydroxyphenyl)-1,3,4-thiadiazole
(TS), and 2-amino-5-(2-hydroxy-5-sulfobenzoyl)-1,3,4-thiadiazole (TSF) (Scheme 1A–F).
It is also noteworthy that the aforementioned 1,3,4-thiadiazole analogues show, apart from
promising biological properties, also rather interesting spectroscopic properties. The same include
the atypical effect of the tautomeric shift induced by changes in medium polarizability (changes of
the keto/enol equilibrium [
changes in the fluidity dynamics of lipid membranes [
spectral forms (such as monomers/aggregates) in micellar systems [
5
]), effects related to crystal polymorphism [
], or the effects of the emergence of various
]. Associating the aforementioned
6] and solvatomorphism [7],
8
9
spectroscopic effects with changes of the photophysical/structural properties of said molecules can
significantly contribute to our understanding of the phenomena underlying the pharmacological
properties of the analyzed analogues.
However, the primary focus of the presented study was on spectroscopic analyses and
quantum-mechanical calculations aimed at understanding the compounds’ unique fluorescence
properties related to the effect of dual fluorescence emission. The analogues selected for the study
(Scheme 1A–F) were analyzed in aqueous solutions with varying pH, supported by studies in selected
organic solvents (for easier identification of aggregation effects and changes in the dipole moments
between ground and excited states). The paper aims to explain the observed fluorescence effects
related to the registered—in low pH conditions (as well as concentration dependent in solvents such as
butan-1-ol)—effect of dual fluorescence TS or the emergence of several fluorescence bands (depending
on the pH or the compound’s concentration). The study identified a correlation between the observed
effects and a specific molecular form of the analyzed fluorophore for a given pH. The effects are
particularly interesting as the selected analogues do not significantly vary in terms of the structure of
their respective chromophoric systems. The most notable differences between the selected analogues
are observed in the structure of their resorcylic substituent groups (Scheme 1A,C,E; not studied to date
for this particular analogue group), which has not been subject to research in this type of media to date.
By employing spectroscopic methods including electronic absorption and fluorescence spectroscopy,
fluorescence lifetime and time-dependent density functional theory (TD-DFT) calculations, as well as
calculations pertaining to dipole moment fluctuations between ground and excited states, we were
able to demonstrate the complexity of the physicochemical processes responsible for the fluorescence
effects (i.e., the emergence of dual fluorescence emission of several bands thereof). The conducted
spectroscopic studies as well as quantum calculations and dipole moment determinations allowed us
to correlate the observed fluoresce effects with the effects of aggregation taking place in the analyzed
systems and influencing the capacity for a molecular charge transfer in said analogues. Furthermore,
in order to confirm their potential medical applicability, the selected analogues were subjected to
preliminary analyses of their antimycotic properties against a selection fungi species, as well as their
antioxidative properties.
It is also noteworthy that in the literature [10], the phenomenon of dual fluorescence has been
associated with the emergence of two separate emission spectra as a result of a single electronic
excitation. The phenomenon can be induced in a variety of ways, including changes in the medium
polarity, pH [11] or temperature [12], as well as aggregation effects. The theories attempting to explain
the aforementioned fluorescence effects include:
•
intermolecular charge transfer (CT) inducing the emergence of specific CT states [13], where
the effect is combined with molecule twisting and twisted intramolecular charge transfer (TICT)
occurs [14,15];
•
emergence of the so-called excited-state intramolecular proton transfer (ESIPT) [16
the molecules;
,17] in
•
•
in the case of additional concentration-induced effects, we are dealing with excimers [18];
one should also mention processes related to breaking the Kasha rule (e.g., as recently described
in detail by Brancato et al. [19]);

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•
a very interesting explanation of these types of effects provided in the literature also involves the
processes related to molecular aggregation (i.e., aggregation induced emission (AIE)) [20,21].
However, it should be noted and emphasized that modeling the activity of fluorophores relative to
the changes in the spectroscopic properties of the analyzed systems, both under conditions of varying
pH and polarity, becomes particularly significant also in terms of designing specific fluorescence probes
which are always in high demand in the context of molecular biology.
In the 1,3,4-thiadiazoles selected for the study, the emergence of fluorescence effects related to
the dual nature of the same is also closely correlated with the structure of the substituent group used
and modified (in our case for the first time) on the side of the resorcylic system (Scheme 1A,C,E).
We observed that modification of this particular part of the substituent may significantly influence
the aggregation properties (i.e., the way in which molecules aggregate) of the selected analogues.
This observation was corroborated both experimentally and in the conducted quantum-mechanical
calculations. Under specific circumstances (e.g., for TS in low pH medium or butan-1-ol), increasing the
compound’s concentration may trigger processes related to a charge transfer between the interacting
molecules. Studies conducted with the use of stationary and time-resolved fluorescence spectroscopy
on the selected analogues from the 1,3,4-thiadiazole group allowed us to observe, in low pH media and
butan-1-ol, that with increasing concentration, dual or multiple fluorescence bands emerged, partially
overlapping (depending on the structure of the substituent group). Such effects were not observed
for the analyzed group of compounds in pH higher than 5 or other solvents used in the study and
significantly varying in terms of polarity. Furthermore, preliminary microbiological studies on the
compounds’ antimycotic properties revealed an interesting correlation between stronger biological
effects and the analogues displaying the more interesting/less typical fluorescence properties.
The primary goal of this work is to attempt to explain and give a brief description of the dual
fluorescence effect observed in the selected analogues from 1,3,4-thiadiazoles including an aqueous
environment with a different concentration of hydrogen ions, and their dependence on the structure of
the substituent group on the resorcyl system side.
The presented paper marks out significant novelties in comparison to our previous work where
selected analogues from 1,3,4-thiadiazoles were investigated mainly towards specific solvent effect
and basic oxidative properties (thin-layer chromatography (TLC)-DPPH method with “dot-blot” test).
In this paper, the authors intend to investigate if similar fluorescence effects will be observed in
a more physiological environment and compare it with oxidative as well as more important antifungal
activities of these molecules.
Uncovering the exact details of the changes in the molecular organization of the selected
compounds in media with varying concentrations of hydrogen ions and varying polarity is significant
in determining their potential for pharmacological and medical applications as well as the directions
of further model studies in media such as micellar o liposomal systems. Such molecules may also be
used as molecular probes very sensitive to environmental changes in systems studies in the context of
molecular biology [22]. For this purpose, molecules such as 1,3,4-thiadiazoles which exhibit the dual
fluorescence emission effect derivatives are in demand.
2. Results and Discussion
The selected compounds from the 1,3,4-thiadiazole group were synthesized in such a way so
as to obtain significant differences in the structure of the substituent group in the resorcylic system
(Scheme 1A,C,E; Scheme 2). In the case of 2-amino-5-phenyl-1,3,4-thiadiazole (TB), the structure
consists solely of the benzene group bound directly to the 1,3,4-thiadiazole ring and an –NH2
group. 2-Amino-5-(2-hydroxyphenyl)-1,3,4-thiadiazole (TS) contains, in its benzene ring, a substituted
hydroxylic group in the ortho position. In the case of 2-amino-5-(2-hydroxy-5-sulfobenzoyl)-
1,3,4-thiadiazole (TSF), the benzene ring includes an –OH group substituted in the ortho position
(analogically to TS). The remaining elements of the molecular structure are the same in all the analogues.

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2.1. Analysis of Spectroscopic Effects
Figure 1 presents the electronic absorption spectra for the 1,3,4-thiadiazoles selected for the
study, at varying concentrations of hydrogen ions in the aqueous solution used, respectively for TB in
Figure 1A, TSF in Figure 1B, and TS in Figure 1C (for clarity of the presentation, spectra for pH 2, 4, 6,
8, 10, and 12 were selected).
0.60
A
0.45
0.30
0.15
TB in H2O
pH = 2
pH = 4
pH = 6
pH = 8
pH = 10
pH = 12
0
B
0.20
TSF in H2O
0.15
0.10
0.05
pH = 2
pH = 4
pH = 6
pH = 8
pH = 10
pH = 12
0
C
0.20
TS in H2O
0.15
0.10
0.05
0
pH = 2
pH = 4
pH = 6
pH = 8
pH = 10
pH = 12
250
300
350
400
Wavelength (nm)
Figure 1. Absorption spectra of thiadiazoles TB, TSF and TS dissolved in H2O at varying pH (from 2
to 12). ( ) TB; ( ) TSF; and ( ) TS. The spectra were not normalized. The absorbance intensity was
A
B
C
adjusted to avoid the reabsorption effects.
The obtained results indicate very clear changes in the shape of the spectra, particularly in the
region significant to physiological values. For the solvents used in the study, the positions of the
absorption and fluorescence maxima as well as the respective Stokes shifts are presented in Table S1 (see
the Supplementary Materials). As clearly visible in Figure 1B,C, the dissociation of the –OH group from
the resorcylic ring in the ortho position (Scheme 1C,E) results in a clear hypsochromic spectral shift by
301 cm−1 for TSF and 303 cm−1 for TS in the case of spectra observed at pH 12, and bathochromic shift
by 1016 cm−1 for TSF and 684 cm−1 for TS in the case of spectra observed at pH 2 (as well as pH 1)
relative to the spectra of the same compounds at pH 7. For TB, Figure 1A reveals only a slight shift of
the bands within the pH range of 6–12 (without the –OH group in the resorcylic system). As evident in
the case of all analyzed analogues, the process of ionization may also be accompanied by the processes
of compound aggregation [23,24]. At pH of approximately 6–8 (depending on the substituent), a very
clear broadening of the absorption spectrum can be observed (mainly) for TS and TSF (the effects are
significantly less prominent for TB) (Figure 1A), indicating the possible presence of forms other than
the monomeric spectral forms of the analyzed structures [9]. In the case of TS and TSF spectra at pH
~2, the spectral absorption is the least pronounced, which may suggest a significant prevalence of
aggregated forms in the case of these analogues (see Figure 1B,C). Meanwhile, no such drastic decrease

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of the absorbance level was observed for TB within the same range and it was significantly higher at
the same pH (compared to TS and TSF). This also clearly evidences the influence of the structure of
compounds selected for the study on the manner in which aggregated molecular structures are formed.
In the case of TB at pH 2, the compound’s absorbance was slightly decreased relative to other spectra
recorded for higher pH, suggesting an increase in the number of the analogue’s monomeric forms.
In the case of TS and TSF, such behavior was not observed in the studied concentration range, which
may confirm the fact that the molecules of those analogues interact more strongly and are therefore
able to form more durable aggregated forms (Scheme 1B,D,E). It is also noteworthy that in the case
of TB at pH 2, the spectrum shifts (primarily) towards the shorter wavelengths, which, according to
Kasha’s exciton splitting theory, may suggest the presence of card pack analogues (the most likely
for this analogue). Moreover, for low pH values it cannot be excluded that the thiadiazole ring is
subject to protonation and an ionic form with the -N+-H group is formed in all the studied analogues.
The effect was most evident in the case of TB but was also present for TS and TSF, although the latter
two analogues containing an –OH group may additionally be subject to head to tail aggregation as
well. This was reflected in the absorption spectra where the long wavelength spectra registered for
this analogue revealed a clear broadening of the bands. In the case of TSF, we observed what most
likely was a greater equilibrium between the respective types of aggregation together with molecule
ionization, whereas for TS, we recorded a band with the maximum at approx. 350 nm, evidencing the
possible presence of aggregated structures even larger than dimers.
Moreover, in the case of TS a (fairly) clear isosbestic point could be observed at approx. 345 nm,
which may suggest that the transfer between the respective aggregation forms in the compound is
highly fluid (and the molecules are susceptible to such interactions). For the band located in the
long wavelength side, this points to a stronger aggregation (head to tail) whose level is most likely
constant at a low pH. The intensity of bands evidencing the aggregation effects occurring in the
selected analogues is also significantly correlated with the structure of the very substituent group in
the resorcylic ring bound to the 1,3,4-thiadiazole ring.
Figure 2A presents the titration pH-metric curves and pK points for the ionization-related –OH
groups in the resorcylic ring of TS and TB, as well as the pK points for all compounds for the
-N+-H group.
12
14
A
B
12
10
8
10
8
TB
TSF
TS
6
6
4
4
2
2
0
75
150
225
300
375
2
4
6
8
10
12
pH
HCl volume (l)
Figure 2.
(
A
) pH-metric titration curves for TB (black squares), TSF (red circles), and TS (blue triangles).
) The ratio of the maximum electron spectra at c.
Titration was carried out with the use of 1 M HCl. (
B
309/330 nm for TB, TSF, and TS relative to changes in the pH of the aqueous solution.
In the case of TS and TSF, the pK for the –O− group was, respectively, 7.35 and 7.02, and for the
–N+-H group, the pK for all the compounds was approx. 4.85. For pK higher than the larger of the
above, we observed for the respective analogues (TS and TSF) the prevalence of forms with the –OH

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group, whereas for pK below the lower of the above, for each analogue the prevalence of forms with
the –N+-H group was observed. Figure 2B presents the ratio of absorbance intensity at approx. 309–330
nm in all compounds relative to changes in the pH of the aqueous solution. It can be observed that
for pH from 1 to approx. 7, TS and TSF clearly showed a drastic transition between the forms with
the aforementioned groups subject to ionization (-O− and –N+-H). The most significant change was
observed for TSF (for which we could probably observe the greatest equilibrium/transfer between the
respective types of ionized forms that could undergo the given types of aggregation) and the changes
were also relatively clearly manifested for TS. In the case of TB, the changes of this type were the least
extensive, as confirmed by the observations in Figure 1A. The significant reduction of this ratio in the
case of TS and TSF further confirms the occurrence of the aggregation effects discussed above. It also
corroborates the conclusion that the process of nitrogen atom protonation was in equilibrium and did
not affect the intensity of the ratio for the selected 1,3,4-thiadiazole analogues within the pH range of 1
to approx. 5/6.
Figure 3A,B presents example fluorescence emission spectra for TB (Figure 3A) and TSF (Figure 3B)
corresponding to the respective absorption spectra.
300
375
A
B
TSF in H2O
TB in H2O
300
225
150
75
225
150
75
pH = 2 Em (Ex 327)
pH = 2 Em (Ex 282)
pH = 4 Em (Ex 308)
pH = 6 Em (Ex 302)
pH = 8 Em (Ex 292)
pH = 10 Em (Ex 292)
pH = 12 Em (Ex 267)
pH = 12 Em (Ex 293)
pH = 2 Em (Ex 318)
pH = 4 Em (Ex 297)
pH = 6 Em (Ex 295)
pH = 8 Em (Ex 294)
pH = 10 Em (Ex 295)
pH = 12 Em (Ex 295)
0
0
300
375
450
525
375
450
525
Wavelength (nm)
Figure 3. Fluorescence emission spectra of (A) TB and (B) TSF dissolved in H2O at different pH.
The excitation was set at the absorbance maximum of each sample, respectively, as pointed out in the
figure. The spectra were measured at room temperature.
The excitation wavelength for all the analyzed samples corresponds to the absorption spectra
maximum (Figure 1A,B). In the case of the fluorescence emission spectra in TB, we observed virtually
no shifts relative to changes in the solution pH or excitation wavelength (at the maximum characteristic
of monomeric or aggregated forms), only significant, pH-dependent changes in intensity (the intensity
of fluorescence decreased with decreasing solution pH). In Figure 3B, presenting the fluorescence
emission spectra for TSF, we observed both a slight shift of the fluorescence emission bands correlating
to the pH of the analyzed solution, and changes in emission intensity as well as a noticeable change
in the band shape depending on the excitation wavelength, which confirms the possible presence of
both respective ionized forms and specific spectral forms (i.e., monomeric or aggregated forms or
for example, dimers). We can therefore clearly observe the influence on the location of the emission
band maximum for the given compound relative to the structure of the substituent group. However,
in all the presented fluorescence emission spectra, a single fluorescence band was observed with the
maximum at approx. 382 nm (TB) or 405 nm and approx. 415 nm (for TSF). It is noteworthy that
in the case of the selected analogues, changing the concentration of hydrogen ions in the medium
has a significant impact on the stability of the excited state of the given molecule in the particular

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solvent. It is noteworthy that in the case of selected analogues TB and TSF the emission observed is
predominant in their monomeric forms.
However, the most interesting effects observed in the presented study were those presented in
Figure 4A–F.
pH = 4
pH = 2
A
C
E
B
60
40
20
Em (Ex 290)
Em (Ex 309)
Em (Ex 320)
Em (Ex 340)
Em (Ex 290)
Em (Ex 310)
Em (Ex 320)
Em (Ex 340)
600
400
200
0
0
pH = 6
D
pH = 8
Em (Ex 275)
Em (Ex 298)
Em (Ex 314)
Em (Ex 266)
Em (Ex 292)
400
400
200
200
0
0
pH = 10
pH = 12
F
Em (Ex 265)
Em (Ex 292)
Em (Ex 314)
Em (Ex 263)
Em (Ex 293)
Em (Ex 307)
400
400
200
0
200
0
400
500
400
500
Wavelenght (nm)
Figure 4. Fluorescence emission spectra of TS dissolved in H2O at different pH: (
A
) pH = 2, (
B
) pH = 4,
(
C
) pH = 6, ( ) pH = 8, ( ) pH = 10, and (F) pH = 12. The excitation was set at the absorbance
D
E
maximum of each sample, respectively, as pointed out in the figure. The spectra were measured at
room temperature.
In order to provide a further, more in-depth insight into the character of the molecular interactions
occurring for TS in aqueous solutions of varying pH (at pH levels corresponding to the results in
Figure 1C), its respective emission spectra were presented for various excitation values. As can
be clearly noticed, for different excitation wavelengths (corresponding to the absorption spectra in
Figure 1) we observed the emergence of several fluorescence emission bands, shifted relative to each
other, or the effect of dual fluorescence in the case of pH 2 and 4 (grey line in Figure 4A,B) for the
excitation wavelength corresponding to the aggregated form, such as a dimer (in the head to tail layout).
For all the presented spectra, the excitation wavelength used corresponded to the prevalence of the
monomeric form (short-wave excitation at the wavelength of approx. 290 nm) and the prevalence of
the aggregated form (excitation in the long wavelength side of the spectrum). Figure 4A,B presents
the emission spectra for TS at the pH of 2 and 4, respectively. Noticeably, after excitation with the
wavelength corresponding to the maximum of the emission spectrum and the maximum characteristic
of aggregated forms, the emission spectrum produced a clearly visible dual fluorescence effect (grey
line). For pH higher than 5, depending on the length of the excitation wave, we observed clear shifts

Int. J. Mol. Sci. 2019, 20, 5494
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of the fluorescence emission spectra but with only single emission bands. It should be pointed out,
however, that said bands overlapped to a considerable extent and their emission maxima corresponded
to the first or second emission band from Figure 4A,B. One should also mention the very clearly
visible correlation between the varying locations of the emission bands and the excitation wavelengths
applied. Depending on the excitation wavelength used (on the short wavelength side approx. 290 nm
or on the long wavelength side approx. 320 nm) aggregated forms of a specific type were more
commonly excited, respectively card pack or head to tail forms (as further confirmed in the TD-DFT
calculations). Naturally, the emergence of the respective aggregation forms was, in this case, closely
related to the particular solution pH, which will be further discussed in the following part of this paper.
The technique of resonance light scattering (RLS) allows a highly probable identification of the effects
related to the given type of chromophoric aggregation of the studied analogues [25], as well as the
differing character of such aggregation interactions.
Based on the exciton splitting theory and by employing quantum-mechanical TD-DFT calculations,
we were able to conclude that the emergence of the very interesting and atypical spectral shifts in the
emission spectra registered for TSF and, most notably, the effect of dual fluorescence observed for TS
in the pH range of 1–4, was significantly influenced by the presence of different aggregation forms,
specifically card pack aggregation (more characteristic for short wavelength excitation) and head to tail
aggregation (characteristic for long wavelength excitation) of the analyzed molecules. Furthermore,
by using the spectral shift in the absorption spectra in combination with the mechanism provided by
the exciton splitting theory, we were also able to calculate the distances between adjacent chromophores
of the analyzed molecules [23,26]. So, for the potential TB dimers, the distance between adjacent
chromophores obtained in the calculations was approx. ~3.67 Å, whereas for TSF that distance was
approx. ~3.61 Å [26]. In the case of TS, the distance was the smallest at approx. ~3.52 Å. Thus, in the
case of TS and TSF, aggregational interactions very strongly influence the observed fluorescence effects.
To recapitulate at this point, one should strongly emphasize the impact on the observed spectral
effects (in particular in the emission spectra of TS and TSF) of such factors as, above all, the phenomenon
of molecular aggregation (of two types) and difference in the substituent structure of the respective
compounds (which naturally partially correlates with the former of the two factors).
We can therefore preliminarily posit that the fluorescence emission spectra of the analyzed
compounds under high pH conditions are dominated by the emission from monomeric forms; various
ionized forms, and at low pH, in the case of TS/TSF, form the two aforementioned aggregated forms. For
TS, at pH lower than 5, the aggregation processes may, given the prevalence of card pack aggregative
interactions, also trigger effects related to intermolecular charge transfer (CT), which influence the dual
character of emission spectra, as confirmed in the conducted TD-DFT calculations as well as, most
importantly, calculations of the dipole moment in the ground and excited state (described below).
Next, Figure S1A,B presents the fluorescence excitation spectra (Ex) for TB (Figure S1A) and TSF
(Figure S1B), corresponding to the fluorescence emission spectra in Figure 3A,B.
Emission in the case of excitation was registered at the wavelengths corresponding to the maxima
of the respective fluorescence emission spectra in Figure 3A,B. The much higher selectivity of the
fluorescence excitation spectra compared to the electronic absorption spectra facilitates the excitation
of a specific molecular form or compound in the analyzed analogue. In other words, the fluorescence
excitation spectra allow the excitation of a specific spectral form of the compound (e.g., the monomer,
dimer, etc.). Figure S1A presents the fluorescence excitation spectra for TB at the selected pH levels
(corresponding to the results discussed above). It can be observed that in the case of TB, virtually
only the band characteristic for the compound’s monomeric forms was observed (varying in intensity
depending on the pH). However, said bands were quite significantly widened, which suggests that
even in the case of this analogue the effects of aggregative interactions may be observed. Figure S1B for
TSF presents, apart from the band with the maximum at approx. 300 nm, a very clearly visible band on
the long wavelength side with the maximum at approx. 360 nm, varying in intensity depending on
the excitation wavelength (short or long wave excitation, as described above) and pH, characteristic

Int. J. Mol. Sci. 2019, 20, 5494
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for aggregated forms emerging in correlation with the presence of this particular ionic form of the
compound. So, in the case of TSF we observed the highest intensity of fluorescence excitation bands in
the region associated with aggregation (head to tail, long wave). As already mentioned, the fluorescence
emission spectra for TSF revealed, in correlation with the solution pH, bands that were partially
overlapping and shifted relative to each other.
For TS, Figure S2 presents excitation spectra analogical to those observed in TB and TSF and
presented in Figure S1, at varying pH levels of the aqueous solution (analogically to the emission
spectra in Figure 4). Similarly, to TSF, in the case of TS we observed, depending on the pH and
excitation wavelength, band enhancement on the long wave side (which evidences the prevalence of
card pack aggregation). As visible in the excitation spectra, bands originating from the aggregated
forms are more strongly evidenced for pH levels at which the emissions spectra showed effects related
to dual fluorescence, i.e. pH 2 or 4.
Within these pH ranges, the spectra show a significantly increased half-value width relative to
other pH levels, which clearly suggests the presence of other than monomeric forms of the compound.
Thus, for TS (and to a considerable extent also TSF) we can observe the strongest effects related to
exciton splitting at the main energetic level S0, which induces the respective changes in the fluorescence
emission spectra. In the pH ranges where the effects of dual fluorescence or multiple bands significantly
shifted relative to each other are not observed, the excitation spectra at wavelengths corresponding to
the respective forms of the analyzed compounds are fairly consistently overlapping within practically
the entire wavelength range.
To emphasize the influence of the type of aggregation on the observed fluorescence effects, one
needs only to carefully analyze the results presented in Figure 5.
275
300
325
350
pH = 2
375
275
300
325
350
pH = 3
375
0.9
0.6
0.3
Ex (Em 410)
1-T
Ex (Em 418)
1-T
0.3
0.0
pH = 2
Ex-(1-T)
-0.3
-0.6
pH = 3
Ex-(1-T)
pH = 4
pH = 5
0.9
0.6
0.3
Ex (Em 422)
1-T
Ex (Em 411)
1-T
0.3
0.0
pH = 4
Ex-(1-T)
pH = 5
Ex-(1-T)
-0.3
-0.6
pH = 6
pH = 7
0.9
0.6
0.3
Ex (Em 410)
1-T
Ex (Em 410)
1-T
0.3
0.0
pH = 7
Ex-(1-T)
pH = 6
-0.3
-0.6
Ex-(1-T)
275
300
325
350
375
275
300
325
350
375
Wavelength (nm)
Figure 5. Fluorescence excitation spectra normalized at the maximum (to the value of 1) in comparison
with 1-T spectra (T: transmission) of TS dissolved in H2O at different pH (from 2 to 7). Below,
the respective differential spectra Ex-(1-T) corresponds to the spectra in the panels above.

Int. J. Mol. Sci. 2019, 20, 5494
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The figure illustrates the overlap between the normalized excitation spectra (presented in Figure S2
for TS) and the 1-T spectra for this compound (T: transmission) and the differential spectra under each
respective panel (Ex-(1-T)). Such a presentation of the spectra allows us to observe the impact that
a certain type of aggregation has on the aforementioned fluorescence effects. For the pH levels at
which the effect of dual fluorescence is present, we observed band enhancement (in the differential
spectra) on the long wave side (characteristic of head to tail aggregation) (e.g., for pH 2–5). Whereas,
with increasing pH (at 6 two types of aggregation are evident and the enhancement is present on both
sides), already at the level of pH 6, we observed increased enhancement on the short wave side, which
implies the prevalence of head to tail aggregation, and in the emission spectra, the disappearance of
effects related to dual fluorescence emission.
To recapitulate, based on the theory of exciton splitting, the characteristic long wave bands should,
in the case of the analyzed analogues, be associated with various types of aggregated forms, as further
confirmed by the RLS spectra (presented in Figure 6 and Figure SA3).
400
TB
A
300
200
100
0
150
100
50
pH = 2
pH = 4
pH = 6
pH = 8
pH = 10
pH = 12
TSF
B
0
200
150
100
50
TS
C
0
300
400
500
600
Wavelength (nm)
Figure 6. Resonance light scattering spectra (RLS) of (
different pH.
A) TB, (B) TSF, and (C) TS dissolved in H2O in
According to the exciton splitting theory, the observed shifts are related to the prevalence of card
pack aggregation, long wave enhancement, and in TB with head to tail aggregation [23]. Based on
the above, it can be inferred that the observed fluorescence effects are certainly related to the effect of
molecular aggregation taking place in the used solutions of varying pH. In the case of TS, the card pack
aggregation facilitates the emergence of intermolecular CT states (as confirmed by dipole moment
calculations discussed later in the text), whereas in TSF molecules, due to the structure of its substituent
group, despite the most likely very heavy card pack aggregation the preference is also for head to tail
aggregative interactions and a charge shift evidenced by the split of the fluorescence emission spectra
into two bands characteristic of the respective electronic states.

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As the subsequent research step, in order to confirm one of the main (and necessary)
factors influencing the effects observed in the fluorescence spectra (i.e., aggregation), spectral RLS
measurements were conducted (∆λ = 0) for the selected derivatives in the entire pH range. Figure 6
presents the RLS spectra for TB (Figure 6A), TSF (Figure 6B), and TS (Figure 6C). As provided in the
literature, the occurrence of RLS spectra should usually be associated with chromophoric aggregation
of the interacting molecular systems [25]. It is clearly apparent that the RLS spectra confirm the above
assumption related to the significant impact of aggregation effects on the changes in fluorescence
spectra observed above. Figure 6A illustrates the presence of RLS bands for TB in practically the entire
pH range; however, the spectra (except only high pH in the range of 10–12) only slightly differ from
each other, similarly to fluorescence emission spectra for this compound relative to the changing pH.
Figure 6B presents the RLS spectra for TSF at selected pH levels; the tendency here is very similar to
that observed for TB, however, in this case the shifts recorded in the emission spectra are considerably
more significant (and the shape of RLS spectra for TSF is considerably different than in the case of
TB, which suggests the presence of different aggregation forms). In Figure 6C, similarly to the other
analogues (presented in Figure 6A,B), RLS spectra are also clearly present in practically the entire
range of hydrogen ion concentrations (slightly less prominent at high pH). One should emphasize at
this point that RLS spectra were observed for all the analyzed analogues, irrespective of the changes
observed previously in the emission fluorescence spectra. Within the wavelength range of 350 to
420 nm, we registered the most significant changes in RLS scattering for the studied analogues, which
corroborates the significant participation of head to tail aggregation in the observed aggregative effects,
as already discussed in the context of absorption and fluorescence excitation spectra. The effects are
very evident in Figure S3, representing the ratio of RLS scattering intensity at the wavelength of approx.
436 nm relative to pH, where one can directly observe that RLS spectra are present practically in the
entire range of pH levels.
In summary, it is noteworthy at this point that the RLS spectra related to chromophoric aggregation
of the analyzed molecules are very characteristic and clearly correlate the observed fluorescence effects
with various types of said aggregation. However, it should also be emphasized that the same is not
the only factor determining the observed fluorescence effects as very intensive RLS bands were also
observed for TB where, as discussed in the context of previous experiments, we observed no significant
changes in the fluorescence emission spectra relative to the emergence of dual fluorescence or presence
of multiple fluorescence emission bands partially shifted relative to each other. The oscillative structure
of the observed RLS bands evidences the abundance of aggregated structures of varying sizes in
the analyzed compounds [25] and may confirm our earlier assumptions regarding the presence of
different aggregated forms of the analyzed molecules. In the pH range in which a greater aggregation
tendency of molecules is observed, the effects of broadening the emission band or its significant shift
or other dual fluorescence emissions-related effects are observed much faster. Both the excitation and
RLS spectra explicitly relate these two factors together. The aggregation process with appropriate
molecular structure (and above all the application of its conformation) can cause changes in fluorescence
emission spectra.
Nonetheless, it can be concluded with considerable certainty that in the case of the observed
fluorescence effects, aggregation is a factor necessary for their emergence. The above hypothesis is
corroborated by the results presented in Figure 7A–F as well as in Figure S4A,B.
Figure 7A–C presents the fluorescence emission spectra for TS at three different excitation
wavelengths (248 nm, 313 nm, and 345 nm, respectively) relative to the concentration of the analyzed
compound. It can be observed that regardless of the length of the excitation wave, with increasing
compound concentration the emission also grows significantly at approx. 435 nm in the dual
fluorescence spectrum. This clearly evidences the impact of aggregation effects on the studied
fluorescence effects. An analogical experiment was conducted for the TSF derivative (i.e., one involving
three excitation wavelengths and variable compound concentrations). We observed, firstly, that the
bands with the maximum at 413 nm (Figure 7F) and 433 nm (Figure 7E) showed a significant loss in

Int. J. Mol. Sci. 2019, 20, 5494
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intensity with the increasing concentration but nonetheless remained present. Secondly, even for TSF
(where spectra measured for varying pH did not show this effect) we observed a clear effect of dual
fluorescence in Figure 7D under short wave excitation (e.g., sample 5). Furthermore, it is important
to notice that more intensive short-wave emission was observed under short wave excitation in the
region characteristic of head to tail aggregation enhancing the card pack aggregation, which is clearly
visible in Figure S4 (black dots on both panels).
Figure 7. Fluorescence emission spectra of TS and TSF dissolved in butan-1-ol. For TS: (
at 284 nm; ( ) excitation at 313 nm; and ( ) excitation at 345 nm. For TSF: ( ) excitation at 278 nm;
) excitation at 317 nm; and ( ) excitation at 335 nm. Measurements were carried out for six different
A) excitation
B
C
D
(
E
F
dilutions: sample 0: 3 mg of TS/TSF dissolved in 2 mL of butan-1-ol; sample 1: sample 0 + 1 mL of
butan-1-ol; sample 2: sample 1 abstract 1 mL + 1 mL of butan-1-ol; sample 3: sample 2 abstract 1 mL +
1 mL of butan-1-ol; sample 4: sample 3 abstract 1 mL + 1 mL of butan-1-ol; sample 5: sample 4 abstract
1 mL + 1 mL of butan-1-ol.
In order to better understand the mechanism of the spectral shifts described above (Figure 1,
Figure 3, Figure 4) as well as the fluorescence effects observed in the compounds, as shown in Figure S5
and Figure 6A, we presented the correlation between Stokes electronic transition π→π* and the
changes in the function of polarity fluctuations ENT as well as F(
polarity/polarizability of the solvent used.
ε,n) which describe changes in the
Firstly, as can be observed for all selected compounds (Figure S5, Figure S5A), the slopes of the
lines are positive for all the solvents used, which usually signifies an increase in the dipole moment of
the excited molecule and may suggest that its direction is preserved during the electron transition.
This fairly clearly evidences the possibility of CT between the molecules triggered by aggregation
interactions, causing the particular observed fluorescence effects, such as dual emission in the case of TS.
Furthermore, it confirms the significant impact of aggregation effects on the non-specific interactions
taking place between the molecules of the analyzed compounds. Furthermore, the somewhat irregular

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linear dependencies observed in the presented relationships (Figure S6B) largely confirm the possibility
of triggering aggregative interactions by hydrogen bonds, particularly in environments where dual
fluorescence emission or two separate, largely overlapping, emission bands are observed. The presence
of solvent molecules modifies the absorption and fluorescence spectra of the analyzed compounds,
usually leading to shifts in the locations of their maxima towards longer, less frequently shorter
wavelengths. However, the presence of hydrogen bonds (triggering aggregation which may influence
CT-related effects) may lead to significant departures from that model [27]. Furthermore, one should
also consider the fact that in systems such as these studied here, we observe a continuous equilibrium
between the formation of intermolecular and intramolecular hydrogen bonds. The former lead to
the formation of aggregated systems (firstly of the dimer type), as clearly evidenced by the changing
intensity of RLS spectra and fluorescence excitation spectra.
Most notably, however, one should take into account the results of dipole moment calculations
performed for the analyzed molecules in ground and excited states based on their respective absorption
and fluorescence emission spectra in correlation with Reichardt’s [28] and Kawski’s methods [29],
respectively, Equation (2) and Equations (5)–(7). The most important factors pertinent to those
calculations are presented in Table 1 for the former and Table 2 for the latter method, for comparison
and corroboration of the results.
Table 1. Slope m from Stokes shift versus microscopic polarity function, change in dipole moment ∆µ
,
and coefficient of determination r2 for TB, TS, and TSF.
Compound
m
∆µ (D)
r2
TB
TS
TSF
3554.36
8976.70
8729.69
2.37
3.75
4.00
0.82
0.91
0.89
Table 2. m1 and m2 determined by Stokes shift against function F(
ε
,n), and m2 determined by (va
+
vf
)
against F(ε,n) + 2g(n), ground state µg, excited state µe, change in dipole moment ∆µ, and correlations
factor r2 determined for both figures for compounds TB, TS, and TSF.
µe
µg
cm3
Compound
m1
m2
µe (D) a
µg (D) b
∆µ (D) c
r2
a0 [Å]
Vm
[
]
mol
TB
TS
TSF
132.9 ± 3.0
131.3 ± 3.0
154.1± 3.0
3.75
3.73
3.94
1852.26
3748.56
3948.56
−9217.88
−18,746.02
−16,703.42
1.50
1.50
1.62
9.30
13.21
12.80
6.19
8.81
7.91
3.11
4.40
4.90
0.76:0.75
0.91:0.95
0.82:0.95
1 D = 10−18 statC
·
cm; a Value calculated from the plot of Stokes shift vs. F(
ε
,n), b Value calculated from the plot of
c
va + vf shift vs. F(ε,n) + 2g(n). The dipole change calculated from the experimental values of µg and µe.
As we can see, the most significant changes of the dipole moment of the analyzed molecules
(between the ground and excited states) were observed for TS molecules and TSF, the compounds
whose fluorescence emission spectra revealed the most interesting fluorescence effects (depending
on the excitation, either two largely overlapping fluorescence emission bands or dual fluorescence).
The above calculations confirmed in this case that CT occurred between the analyzed molecules. In the
case of TB and TS we observed the ratio in the dipole moment between the excited and ground states
of 1.50 whereas in the case of TSF the difference increased to 1.62 (D). Results of the above calculations
confirm the significant influence of the structure of the substituent system on the observed fluorescence
effects. They also suggest that an important effect of solvatochromic shifts entails the intersection
of states with varying distributions of electronic density dictated by the polarity of the respective
medium. In polar solvents, electronic states with high dipole moments (often in fact the CT states) are
stabilized relative to the states with low dipole moments. If such states are characterized by similar
energies, a change in the polarity/polarizability of the medium may lead to a reversal of the states’
order. Since photochemical properties are determined by the character of the lowest excited state of
the given multiplicity (Kasha’s rule), they can be (as observed in the present study) subject to very
significant changes with the solvent’s polarity.

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2.2. Fluorescence Lifetime Analysis
In the subsequent part of the study on the mechanism of molecular interactions inducing the
respective fluorescence effects in the emission spectra, as presented above in Table S3 (and Figure S7),
we considered the results of time-resolved measurements of fluorescence lifetimes conducted for all
analyzed analogues in the full range of pH levels.
The methodology is presented and described in detail in Section 3 (Section 3.5). Fluorescence
decay for TSF was single-exponential. The measured fluorescence lifetime was between 0.37 and
5.43 ns, depending on the pH of the solvent (the correlation is presented in Figure S7). A clearly visible
reduction of the fluorescence lifetime was observed for pH < 4.
The correlation between fluorescence decay and pH observed for TS is similar to that obtained
for TSF. For pH <4, we recorded a significant reduction of the fluorescence lifetime. Moreover, for
this analogue we observed two exponential fluorescence decay at pH = 2, which suggests the possible
presence of more than one molecular form in the solution. The measured fluorescence lifetime for the
main component, 1.64 ns, fits in well with the observed decrease of the parameter at low pH levels,
with the share of this component estimated at 72%. The fluorescence lifetime recorded for the second
component was considerably shorter (0.29 ns). At pH within the range of 4–12, the fluorescence lifetime
for the TS analogue was decisively shorter than in the case of TSF but similarly to the latter, it remained
at a roughly constant level.
In the entire range of pH values, the fluorescence lifetime recorded for TB was the shortest among
all three analogues (0.1–0.18). In the case of this analogue, the second constituent, with the fluorescence
decay time of 1.2 ns and relative share in total fluorescence estimated as 11%–20%, was observed
both at low (pH = 2) and high (pH = 11,12) pH levels. Clearly, aggregation which triggers the effects
associated with dual fluorescence takes place in TS and TSF in practically the whole range of pH values
(the lifetime remains at a fairly constant level). For TB, at pH lower than 7, we observed a far more
prominent presence of the second constituent in the overall lifetime, which indicates the existence of
spectral forms other than aggregates. The reasonability of using fluorescence lifetime measurements in
this paper is worth emphasizing. In particular, due to the process related CT, a significant increase in
the average fluorescence lifetime is observed. Hence, the effect of dual fluorescence reported in this
work, allowed for the discovery of noticeable changes in the fluorescence lifetime of the fluorophore,
which together with noticeable aggregation processes, fully confirms our hypotheses.
To recapitulate the deliberations so far, it should be emphasized that (as largely corroborated by
the results of quantum-mechanical calculations) effects observed in the case of TS and TSF molecules
(in high concentrations) are triggered primarily by the intermolecular phenomenon of charge transfer
(CT) enforced (or facilitated) in this particular case by the phenomenon of molecular aggregation of
a given type (e.g., head to tail or card pack). Also, the structure of the substituent group on the side of
the resorcylic system significantly influences the described overlap of two phenomena.
Figure 8 (and Table 3) provides a schematic, structural presentation of the TS− molecule, TSH
molecule, TSH2+ and sample TS dimer as optimized using B3LYP/aug-cc-pVDZ and polarizable
continuum model (PCM) with water used as the solvent. Table 4 summaries the main predictions
regarding the excited state energetics of these species.
The main computational findings are as follows. Firstly, the TS molecule in the form of TS−, TSH,
and TSH2+ has a single, low-lying and intensive excited state responsible for the shape of its absorption
and emission spectra (Table 3, Figure 8A–C). However, in the case of the TSH molecule, the situation
changes drastically. The TS molecule may form a hydrogen bond both within its own structure, which
in itself affects its spectroscopic properties, as well as with the molecules of the medium. Hence the
single intensive TS state is split in two in this case. This results in a more complex, dual structure of the
TS absorption spectra, as observed in Figure 1, where the absorption spectra reveal band widening on
the long wave side. The subsequent analyzed TS dimer, (TSH)2 is only one of many potentially possible
types of aggregates (as already discussed and described above in the context of the experiments),
therefore the results should be treated as only semi-quantitative (although significant as a theoretical

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confirmation of the experimental results). Nonetheless, the calculations offer a certain insight into the
experimentally analyzed photophysics. As shown in Figure 8, the structure of the TS dimer includes an
intermolecular hydrogen bond, apart from the possible intramolecular hydrogen bonds characteristic
of monomers. The interaction between monomers splits the two excited states of the TS molecule
into four dimer states which transfer intensity onto the respective bands present in the fluorescence
emission spectra (Figures 3 and 4). This fact has a significant bearing on the absorption spectra and
may in fact be key to the overall fluorescence behavior. In one of the discussed dimeric states the
energy of vertical emission is very strongly shifted towards red (bolded in Table 3), which may account
for the observed dual fluorescence; the high energy band is monomeric whereas the low energy band
is dimeric.
A
B
C
D
Figure 8. Ground state structures of TS−
(A), TSH (B (C) and (TSH)2 (D) as optimized using
), TSH2+
B3LYP/aug-cc-pVDZ and polarizable continuum model (PCM) with water as the solvent.

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Table 3. Predictions for low-lying excited states of various forms of the TS molecule in water. Energetics
given in nm scale, dimensionless oscillatory strengths for the absorption process given in parentheses,
SS approach-based energies given in italics. Intense absorbing states and the lowest (emitting)
state reported.
Form
TS−
Vertical Absorption
Adiabatic Absorption/Emission
417/389
Vertical Emission
387/367 (0.38)
283/279 (0.22)
334/315 (0.64)
290/285 (0.17)
326/307 (0.325)
283/279 (0.29)
331 (0.34)
451/421
TSH
363/337
355/330
382
397/368
377/348
449
+
TSH2
(TSH)2
327 (0.22)
323 (0.31)
286 (0.28)
Table 4. Potential of the tested derivatives and selected reference compounds 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid expressed as TROLOX equivalents (TE) (
µ
M antioxidant/
µM
TROLOX) and as IC50 values. Results obtained after 30 min of DPPH• (200
reaction at 25 ◦C. QUER: quercetin.
µM) radicals reduction
TE ± (SD)
IC50 DPPH• (µM) ± (SD)
TB
TS
TSF
QUER
TROL
0.03 ± 0.00
1.10 ± 0.10
0.90 ± 0.07
0.84 ± 0.06
1.00 ± 0.09
1094.64 ± 14.8
34.74 ± 0.29
43.62 ± 0.40
45.83 ± 0.43
41.76 ± 0.38
Also, it is worth mentioning that the dual fluorescence observed in the selected 1,3,4-thiadiazole
analogues may occur via a pathway other than that proposed in this work (two independent modes of
action may be possible). Based on the DFT calculations, the investigated compounds in their ground
state occur solely as enol tautomers. Excitation of the enol form would likely enable an additional and
independent process resulting in dual fluorescence emission. Once in the excited state either cis or
trans-enol the enol form may tautomerize to the excited keto-form, which in turn may emit the long
wavelength fluorescence.
Since the high concentrations are characteristic of aggregation-related dual fluorescence effects,
this second and independent process may be dominant at low concentrations. The possibility of such
mechanism is justified by the lack of clear differences between absorption spectra of the investigated
derivatives recorded in series of various organic solvents. Moreover, according to our preliminary
calculations, the energy differences between the excited enol and excited keto form are relatively small,
which is consistent with the proposed relaxation pathway.
In a conclusion, the hypothetical second mechanism is proposed on the basis of preliminary
calculations and relies upon an excitation of the neutral TS molecule, which in its ground state occurs
as enol tautomer. The excitation of the enolic TS results either in straightforward fluorescence emission
from the excited enol tautomer (higher energy emission band) or the excited keto tautomer is formed
as result of proton (hydrogen) transfer from the phenolic –OH to the nearest thiadiazole nitrogen atom.
The excited state of such keto tautomer is energetically similar to that of the excited enol and may emit
photons manifesting as the second (lower energy) emission band, with subsequent return to the enolic
form characteristic of the ground state.
The proton transfer into one of the nitrogen atoms is favored by the conformation in which the
phenolic –OH resides near the N rather than S atom of the thiadiazole ring. Based on crystal structures
reported to date [7], such conformation is highly likely.

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2.3. Antioxidative Properties of the Analyzed Compounds
The determination of antioxidative potential was performed with a standard spectrophotometric
test with the use of a solution of DPPH• radicals [30
32]. DPPH is a relatively stable radical
which, when dissolved in alcohol, produces a solution with a strong violet color with the absorption
maximum at 519 nm. In the presence of a compound with potential antioxidative properties,
–
λ
max
it undergoes reduction which results in the decay of the absorption maximum at λmax 519 nm and color
change from violet to yellow. The changes are monitored spectrophotometrically. The antioxidative
capacity of a given compound is determined by the speed at which the given analyte solution of
known concentration reduces the fixed concentration of the radical solution. The obtained results
are compared to the reference solution of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(TROLOX). The method entailing the reduction of DPPH• was chosen due to the fact that the studied
thiadiazole derivatives showed no absorption in the wavelength range within which the measurements
are conducted. The obtained, averaged results yielded the curve of decreasing intensity of DPPH•
radical absorption at λmax 519 nm relative to the increasing concentration of the respective analytes
(Figure S8). The inclination angle of the decreasing absorption maximum curves indicates the speed
of the reaction. In the respective points, the values of standard deviation (SD) were additionally
established after three replicates of each experiment.
When comparing the obtained results, one should note the concentration ranges of the respective
compounds necessary to reduce the fixed concentration of radicals. In all cases apart from TB, analytes
concentrated at approx. 60–80 µ
M yielded a reduction of the fixed content of DPPH• radicals by over
70% relative to the initial content thereof under the adopted experimental conditions, whereas in
the case of TB the result was significantly smaller, approx. 7% (Figure S9). The poor activity of the
latter, when compared to TS and TSF, results from its lack of substituent attached to the aromatic ring,
which could take part in the reaction of reducing the radicals. After comparing the results obtained for
the respective derivatives with TROLOX, we obtained the value of antioxidative potential expressed
as a TROLOX equivalent (TE) (Table 4), which specifies how many times higher or lower the given
compound’s potential is compared to the standard. In the case of the analyzed TS and TSF derivatives,
the value oscillated at approximately one, which means that the two analogues are more active than any
of the others including quercetin and the well-known flavonoid. The concentrations of the respective
analytes needed to reduce the DPPH• radicals by 50% of their initial concentration (IC50) (Table 4) to
confirm the above observations.
The lower the concentration required to reduce a fixed number of radicals, the higher the
antioxidative potential of the given compound. In the case of TSF, QUER, and TROL the values are
similar and oscillate within the range of 41–45
µM (for 200
µ
M of the initial DPPH• concentration).
In the case of TS, the value is somewhat lower (34.74
µM), which confirms the higher antioxidative
potential of this derivative (>1 TE). In the case of TB, the value is several times higher than the
concentration of radicals in the analyzed sample, which disqualifies the compound as a potential
antioxidant. In a previous paper all three analyzed analogues of 1,3,4 thiadiazoles were studied for
their antioxidative properties using the TLC-DPPH method with the “dot-blot” test. As expected,
the highest antioxidant activity was observed in TS, while TSF and TB demonstrated lower activity (TS
> TSF > TB, respectively).
Natural compounds such as polyphenols or flavonoids owe their antioxidative properties to the
presence of numerous functional, particularly hydroxyl groups in their structures [33], as the same
can donate the electrons necessary for the reduction to occur. Based on structural analyses of the
studied thiadiazole derivatives it could be preliminarily assumed that the TB derivative, unsubstituted
on the aromatic ring, would show the lowest antioxidative activity, possibly none at all, which was
later corroborated by the conducted experiments. The significantly higher TE and lower IC50 values
(Table 4) observed for TS and TSF are most likely due to the presence in the aromatic rings of those
derivatives of substituents capable of donating electrons.

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2.4. Microbiological Analysis
In addition to the above-mentioned antioxidant properties of the selected studied analogues, it was
decided to pay attention to the potential antifungal properties of some derivatives from this group
of compounds. In the subsequent part of the study, biological tests on selected fungal species were
conducted to determine the antimycotic properties of the analyzed analogues. Candida spp. are the
microorganisms responsible for the most frequently encountered invasive fungal infections in highly
developed countries. The widespread use of azoles and echinocandins has been accompanied by the
emerging drug resistance of clinical isolates of Candida spp. [34]. Moreover, the conventional antifungal
treatments for candidiasis based mainly on polyenes, azoles, and echinocandins are costly and can
cause toxicity as well as side effects [35]. Thus, there is a need to develop novel antimycotic agents.
New pharmaceuticals such as 1,3,4-thiadiazoles can be a good alternative because of their
antibacterial and antifungal properties [2]. The antimycotic potential of 2-amino-5-phenyl-1,3,4-
thiadiazole (TB), 2-amino-5-(2-hydroxyphenyl)-1,3,4-thiadiazole (TS), and 2-amino-5-(2-hydroxy-5-
sulfobenzoyl)-1,3,4-thiadiazole (TSF) was tested in this study against nine Candida species.
The minimum inhibitory concentrations (MICs) of TB and TSF were similar for the tested organisms
and ranged between 128 and >256 µg/mL with the exception of Candida butyri which showed growth
inhibition at 64 µg/mL for TB (Figure 9).
Figure 9. Sample regression curves of selected data showing the effect of examined 1,3,4-thiadiazoles
on Candida species.
The MICs of TS for five Candida species (C. fructus, C. fragicola, C. butyri, C. shehatae, and C. fluviatilis)
ranged between 4 and 64 µg/mL while the other species tested between 128 and >256 µg/mL (Table S4).
Based on the above results and the criteria proposed by Morales et al., TS exhibited strong antimycotic
activity against C. fructus, C. fragicola, C. butyri, C. shehatae, and C. fluviatilis because its MIC values
were lower than 100 µg/mL. An interesting observation is that C. butyri was the most sensitive to

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tested thiadiazole derivatives. Its maximum growth rate was reduced six-fold after the addition of
TS to the growth medium at a concentration of 8 g/mL (Figure 9). At the same time, the doubling
µ
time of the generation was six times longer compared to the control (Table S5). Candida butyri may be
found in humans as opportunistic organisms awaiting the proper change in environmental conditions
to become pathogens, such as in immunocompromised hosts. It was described as the etiological
agent of the candidemia of an immunocompromised cancer patient [36]. Our observations were
consistent with other studies whose results show that Candida aaseri strains (closely related to C. butyri)
were susceptible to such antifungal agents as amphotericin B, flucytosine, ketoconazole, itraconazole,
fluconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and micafungin [36]. However,
new resistant strains continue to be identified and it is therefore important to identify new compounds
with antifungal potential.
3. Materials and Methods
All solvents were of 99% purity or higher (HPLC grade). Thiosemicarbazide, DPPH (2,2-diphenyl-
1-picrylhydrazyl), TROLOX (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), POCl3, benzoic,
salicylic, and 5-sulfosalicylic acids were purchased from Sigma (Darmstadt, Germany). Quercetin,
H2SO4, NH3, and NaOH were purchased from Avantor (Gliwice, Poland).
3.1. Isolation of Thiadiazole Derivatives
TB was synthesized via the method 1, namely the POCl3-catalyzed reaction between benzoic
acid with thiosemicarbazide (Scheme 2) [37]. Benzoic acid was suspended in POCl3 and stirred at
room temperature for 20 min after which an equimolar amount of thiosemicarbazide was added.
The reaction mixture was heated up to 75 ◦C for 6 h and then cooled down to 35 ◦C. Excess POCl3
was quenched by adding water and the reaction mixture was refluxed for another 4 h at 105 ◦C. The
cooled mixture was then brought to pH 8 using a saturated NaOH solution. The precipitate formed
was filtered off, rinsed with water, dried and recrystallized from methanol. An identical procedure
was applied for the synthesis of TS except that salicylic acid was used as the substrate.
TSF was synthesized according to method 2, namely the classical H2SO4 catalyzed route involving
the use of thiosemicarbazide and appropriate 5-sulfosalicylic acid as the substrate (Scheme 2) [38,39].
Scheme 2. Synthetic pathway for the isolation of TB, TS, and TSF.
An equimolar amount of 5-sulfosalicylic acid and thisoemicarbazide was dissolved in concentrated
sulfuric acid and the mixture was heated up to 95 ◦C for 6 h. The mixture was then cooled down and
basified to pH ~8 with use of the diluted ammonia. The excess solvent was removed under vacuum
and the remaining mixture was left undisturbed. The precipitate formed was filtered off, rinsed with
water, and recrystallized from ethanol.
All products were isolated with satisfactory yields and the purities were confirmed by LC-MS
analyses. In particular, experiments comprising of the tandem mass spectrometry (MS/MS) analyses of
the products obtained were performed. The values of the recorded daughter ions were consistent with
those reported in the literature (Figure S10) [40].

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3.2. pH Measurement
The TB, TS, and TSF (about 1 mg) samples were dissolved in 2 mL of distilled water, and then
appropriate aliquots of this solution were added to 50 mL of distilled water. Due to a sparing solubility
of the thiadiazoles tested, the concentrations of samples were not determined. In order to obtain
pH 12, 0.1 M of NaOH was added to each sample. Next, 0.1 M of HCl acid was gradually added
resulting in a desirable pH of aqueous TB, TS, and TSF solutions. The respective titration curve plots
are given in Figure 2. All measurements were carried out at room temperature using the Elmetron
CP-502 pH-meter.
3.3. Measurements of Electronic Absorption and Fluorescence Spectra
The electronic absorption spectra of the selected compounds in selected organic solvents
were recorded using a double-beam UV–Vis spectrophotometer Cary 300 Bio Varian (Middelburg,
The Netherlands) equipped with a thermostatted cuvette holder with a 6
The temperature was controlled with a thermocouple probe (Cary Series II from Varian) placed directly
in the sample.
×
6 multicell Peltier block.
The UV–Vis spectra were recorded on Cary 300 Bio (Varian) instrument. All measurements were
carried out in a thermostatted cuvette holder with a 6
probe (Cary Series II from Varian) was applied for the temperature control.
×
6 multicell Peltier block. The thermocouple
Cary Eclipse spectrofluorometer (Varian) was applied for the recording of fluorescence excitation,
emission, and synchronous spectra. All measurements were carried out at 22 ◦C. All the fluorescence
spectra were recorded with 0.5 nm resolution together with the lamp and photomultiplier spectral
characteristics corrections. Resonance light scattering (RLS) measurements were carried out according
to the previously reported protocol [25,37] with synchronous scanning of both the excitation and
emission monochromators (there was no interval between excitation and emission wavelengths) and
a spectral resolution of 1.5 nm. Grams/AI 8.0 software (Thermo Electron Corporation; Waltham,
Massachusetts, United States) was applied for analysis of the data recorded.
3.4. Methodology and Calculation of Dipole Moments
Analytical grade methanol, DMSO, dimethylformamide (DMF, ethanol, propan-2-ol, butan-1-ol,
acetonitrile, toluene, chloroform, and cyclohexane were used as solvents. The TB, TS, and TSF (about
1 mg) were made up in 1 mL of each solvent. The absorbance intensity was adjusted by the addition of
an appropriate aliquot of TB, TS, and TSF into 2 mL of solvent. The concentration in several samples
remained undetermined due to a poor solubility of the thiadiazoles (TB, TS, and TSF were readily
soluble only in DMSO and DMF). The molar concentrations of TB, TS, and TSF dissolved in DMSO and
DMF were as follows: C1 = 0.056 mM, C2 = 0.055 mM, C3 = 0.038 mM, C4 = 0.062 mM, C5 = 0.056 mM,
C6 = 0.036 mM.
In order to estimate the dipole moments of TB, TS, and TSF molecules, two methods were used
depending on the internal electric field effect.
3.4.1. Method 1
In method 1, the effect of polarization dependence and hydrogen bonding effects with solvents
can be expressed by the function ENT which was first proposed by Reichardt [28,38,39]. A normalized
ET(30) value, specifically the microscopic solvent polarity (E
NT ) is useful in measuring solvent polarity
by solvatochromic methods using betaine dye as a probe solute. ETN is defined by Equation (1) using
water (ENT = 1) and tetramethylsilane (ENT = 0) as the reference solvents:
ET(solvent) − ET(TMS)
ET(water) − ET(TMS)
ET(solvent) − 30.7
ETN
=
=
(1)
32.4

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The change in dipole moment can be determined by Equation (2):
s
81m
∆µ = µe − µg
=
(2)
(6.2/a0)311307.6
where m is the slope obtained from a linear plot of Stokes shift against the microscopic solvent polarity
EN. The values of ET(30) and EN for the solvent are presented in Table S2.
T
T
3.4.2. Method 2
In method 2 the ground and excited state dipole moments were calculated based on Kawski and
Bilot equations [28 29]. By employing Onsager’s reaction field theory and simple quantum mechanical
,
second order perturbation theory of absorption (
they receive the two following relations:
va) and fluorescence (
v
f ) band shift in different solvents,
va − vf = m1F(ε, n) + const
va + vf = −m2F(ε, n) + 2g(n) + const
,n) and F( ,n) + 2g(
(3)
(4)
The expressions giving F(
ε
ε
n
) are provided by Equations (5) and (6). In these
equations, n is the refractive index and ε is the dielectric constants of the solvent, respectively.
!
2n2 + 1 ε − 1 n2 − 1
F(ε, n) =
−
(5)
(6)
n2 + 2
n2 + 2
ε + 2
3
n4 − 1
g(n) =
2
(n2 + 2)
2
Assuming that the symmetry of the investigated solute molecule remains unchanged upon
electronic transition, and the ground and excited-state dipole moment are parallel we obtain the
following equations:


1/2

hca3
m2 − m1


0 

µg
=
(7)




2
2m1

1/2
hca3


m1 + m2


0 

µe =
(8)




2
2m1
m1 + m2
m2 − m1
µe =
µg; (m2 > m1)
(9)
where µg and µe are the ground and excited-state dipole moments of the solute molecule,
constant,
h
is Planck’s
c
is the velocity of light in vacuum, and a0 is the Onsager cavity radius. The value of the
solute cavity radius was calculated based on the Van der Waals volumes (Equation (10)) according to
Edward’s method [40].
4
V = πa30
(10)
3
The calculation of molar volume
m1 and m2 were calculated by plotting Stokes shifts and va + vf against the bulk solvent polarity
functions F( ,n) and F( ,n) + 2g(
), respectively; where, va is the absorption maximum in cm−1 and vf
is the fluorescence maximum in cm−1
V was performed applying ACD/ChemSkech software. Slopes
ε
ε
n
.
The change in the dipole moment can be determined as (Equation (11)):
∆µ = µe − µg
(11)

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The value of the refractive index n, dielectric constant ε of the solvents, functions F(ε,n), and F(ε,n)
+ 2g(n) of the solvents are presented in Table S2.
3.5. Fluorescence Lifetime Measurements
Multifrequency cross-correlation phase and modulation K2 fluorometer (ISS, Champaign, IL,
USA) was applied for the fluorescence lifetime measurements. A continuous wave 280 nm light
emitting diode (LED; model 73115) was applied for the excitation and the decays were recorded in
a 10
×
10 mm quartz cuvette. The fluorescence signal was recorded using a monochromator set at the
10 nm). These emissions were of 405 nm for TSF, 382 nm for TB, and 415 nm
maximum emission (
±
for TS. Measurements were performed for 15 modulation frequencies ranging from 2 to 200 MHz.
The phase shift and demodulation ratios were referenced to the aqueous solution of Ludox@ from
Aldrich (Darmstad, Germany) without the emitter filter. ISS Vinci 2.0 software was used for the data
analysis utilizing a multiexponential decay model with discrete fluorescence lifetime components
(Equation (12))
X
X
fi(λ)
τi
/τi
/τi
I(λ, t) =
αie− t
=
e− t
(12)
i
i
where I(λ,t) is the fluorescence intensity, αi is the preexponential factor, and fi is the fractional
contribution of each fluorescence lifetime component. Minimization of the reduced χ2 value together
with the residual distribution of the experimental data allowed for achieving the best-fit parameters.
The presented results were calculated as an average of three to five repetitions of fluorescence
lifetime measurements.
3.6. Computational Details
The DFT calculations were made using the Gaussian 09 package [41], B3LYP exchange-correlation
functional [42], aug-cc-pVDZ basis set [43], and very dense grids. The solvent effects were modeled
according to the polarizable continuum model (PCM) [44]. Sodium cation and chlorine anion were
used as counterions in the case of charged species. The excited state treatment was carried out using
a standard random-phase approximation (RPA) approach to the TD-DFT formalism [45]. The influence
of the solvent on energetics was determined both according to the standard linear response (LR)
approximation and more exact state-specific (SS) approach [46]. In the case of TS dimer, only the LR
results are provided as the SS procedure was not converging.
3.7. Analysis of Antioxidative Properties
Increasing concentrations of ethanolic solutions of tested thiadiazol derivatives (TB, TS, TSF) were
applied to the following wells of the 96-well plate, so that their final concentration in 200
volume of each sample was within the range of 0–60 M. Samples of reference compounds, namely
quercetin (QUER) and TROLOX (TROL), were prepared in the same way and their final concentrations
in 200 L of the total volume of each sample was within the range of 0–75 M. Immediately before the
measurement, all wells were supplemented with 1 mM of an ethanolic solution of DPPH• radicals,
so that their final concentration in each well was 200 M. The plate was shaken on the internal shaker
µL of the total
µ
µ
µ
µ
of the Tecan Infinite 200 microplate reader (Tecan Austria GmbH, Grödig/Salzburg, Austria) for 10 s to
ensure thorough mixing of reagents in all wells, and spectrophotometric measurements started in the
range of 500–550 nm with a 1 nm wavelength step at 25 ◦C for 30 min. The results obtained were the
average of five exposures of each sample to a beam of light. All experiments were repeated three times.

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3.8. Microbiological Analysis
3.8.1. Antifungal Agents
The antifungal agents (TB, TS, TSF) were dissolved in 100% DMSO, at a final concentration of
g/mL. Ten 2-fold serial dilutions were prepared in Roswell Park Memorial Institute (RPMI)-1640
1000
µ
medium (supports cell growth in research and biomanufacturing was developed by George E. Moore,
Robert E. Gerner, and H. Addison Franklin in 1966 at Roswell Park Memorial Institute, from where it
derives its name.), with the final drug concentrations ranging from 256 to 0.125
agents, in accordance with CLSI guidelines [47].
µg/mL of examined
3.8.2. Fungal Strains and Culture Method
Candida crusei (Polish isolate), C. fructus (JCM 1513), C. fragicola (JCM 1589), C. butyri (JCM 1501),
C. tropicalis (ATCC 1369), C. shehatae (ATCC 22984), C. fluviatilis (CBS 6776), C. freyschussi (CBS 3562),
and C. parapsilopsis (DSM 70125) strains were used in this study. The strains were inoculated onto
Sabouraud dextrose agar (BTL, Poland, Ł
24 h. They were then subcultured on the same medium for a further 24 h at 30 ◦C. The yeast inocula
were prepared by diluting the overnight culture with 0.9% NaCl to 1–5
106 a colony-forming unit
ó
dz´) plates from glycerol stocks and incubated at 30 ◦C for
×
CFU/mL compared to the isolate density standard corresponding to 0.5 in the McFarland scale.
3.8.3. Susceptibility Testing
Thiadiazole derivative MICs were determined using the broth microdilution method described in
CLSI M27-A2 for yeasts. The MIC was defined as the lowest concentration of antifungal agent that
produced a prominent decrease in optical turbidity when compared with the control. The antimicrobial
activity of thiadiazole derivatives was interpreted as strong/good activity (MIC: <100
activity (MIC: 100–500 g/mL), weak activity (MIC: 500–1000 g/mL), and inactive/no antimicrobial
effect (MIC: >1000 µg/mL) according to the criteria proposed by Morales et al. [48].
Fungal inocula (30 L) were added to each well on a sterile, 100-well flat-bottomed microtiter
plate containing the test concentration of thiadiazole derivatives (350 L/well). Each concentration was
µg/mL), moderate
µ
µ
µ
µ
tested in triplicate for each organism. Two wells containing a fungal suspension with no antifungal
agent (compound-free growth control) and two wells containing only growth media (bac◦kground
control) were included in this plate. ODs (optical density) were measured for 24 h at 30 C using
a multi-detection microplate reader (Bioscreen C system, Labsystem, Helsinki, Finland) at 600 nm
and were automatically recorded every 2 h for each well. Turbidimetric growth curves were obtained
depending on the changes in the OD of fungal growth for each antifungal agent concentration and
the compound-free growth control. Growth curve parameters (i.e., max specific growth rate, lag time,
doubling time) were determined using the PYTHON script according to Hoeflinger et al. [49].
4. Conclusions
The fluorescence spectroscopy experiments presented in this paper indicate the emergence in
the fluorescence emission spectra of the selected 1,3,4-thiadiazole analogues (TS) of the effect of dual
fluorescence in varying pH media or for varying concentrations of the analyzed compounds. The effect
studied with the use of fluorescence spectroscopy (with the RLS technique and excitation spectra) and
by measuring the respective fluorescence lifetimes revealed a strong dependence on the phenomenon of
molecular aggregation of the analyzed analogues. A very important role in the context of the observed
fluorescence effects was also played by the structure of the substituent system (in the resorcylic group)
which determined the type of aggregative interactions. The studies of TS fluorescence spectra indicated
the emergence of the dual fluorescence effect for this compound within the pH range of 1 to ~4/5.
Based on the exciton splitting theory, the effects can be associated with the prevalence of card pack
aggregation. Moreover, the calculations pertaining to the changes in dipole moment between the
ground and excited states of the analyzed molecules, performed with two different methods, as well as

Int. J. Mol. Sci. 2019, 20, 5494
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quantum-mechanical TD-DFT calculations very clearly suggest that the possibility of intermolecular
CT may in fact be the main cause of the effects related to dual fluorescence in the case of TS as well as
highly concentrated TSF. The effects of fluorescence emission quenching or the noticeable (albeit slight)
reduction of the mean fluorescence lifetime, confirm the participation of aggregation effects as a factor
necessary for CT to occur. Charge transfers seemed to be particularly evident in the TS molecule, it was
also in this compound that the UV–Vis, fluorescence excitation spectra within the pH range of 1–4/5,
revealed the strongest aggregation effects. Moreover, CT effects must be associated with the correct
conformation of the molecule (i.e., the –OH group in the resorcylic ring), positioned on the side of the
nitrogen atom in the thiadiazole ring, to allow the formation of correct hydrogen bonds.
In case of the investigated 1,3,4-thiadiazole analogues a second mechanism for their dual
fluorescence cannot be excluded. In particular an excitation of the enolic tautomer may lead to the
formation of its excited keto tautomer, and the subsequent fluorescence emission from such excited
keto form of the compound. In order to verify this hypothesis more in-depth studies will be carried
out in the future.
The preliminary microbiological studies also clearly revealed the influence of the substituent
system on the antimycotic properties of the analyzed analogues. The most promising results were
obtained for TS and TSF in which the effects of dual fluorescence were also observed. It is noteworthy
that the studies discussed above may facilitate a quick analysis of the structure of the studied molecule
in any biological or crystalline system, which may allow the whole group of analogues to be used as
excellent fluorescence probes, highly sensitive to changing conditions in the given environment.
Furthermore, one will also be able to exactly determine which molecular form and which particular
conformation is responsible for a given aspect of the biological activity displayed by this group of
analogues. At this point it is also worth emphasizing that due to their interesting photophysical
properties the thiadiazoles selected in this research represent a group of remarkably useful molecular
probes, whose properties easily change depending on environmental conditions.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/21/
5494/s1. Figure S1: Fluorescence excitation spectra of TB (panel A) and TSF (panel B) dissolved in H2O at different
pH. The spectra were measured at room temperature; Figure S2: Fluorescence excitation spectra of TS dissolved in
H2O at different pH (panel a pH = 2, panel B pH = 3, panel C pH = 4, panel D pH = 5, panel E pH = 6, panel F
pH = 7). The spectra were measured at room temperature; Figure S3: Intensity of resonance light scattering spectra
in 436 nm of TB (black circles), TS (red circles) and TSF (blue circles) relative tochange in pH; Figure S4: The ratio
of the maximum fluorescence intensity at 434/492 nm for TS and TSF dissolved in butan-1-ol in different excitation
depending on the changes in concentration; Figure S5: Stokes shift variation with normalized value of solvent
polarity E_TˆN for TB, TS and TSF for various solvent (1—cyclohexane, 2—toluene, 3—chloroform, 4—ethyl acetate,
5—butan-1-ol, 6—propan-2-ol, 7—ethanol, 8—DMF, 9—DMSO, 10—methanol, 11—acetonitrile); Figure S6: Stokes
shift versus F(ε,n) for (panel A), v_a + v_f versus F(ε,n) + 2g(n) (panel B), for TB, TS, TSF dissolved in different
solvents (1—cyclohexane, 2—toluene, 3—chloroform, 4—ethyl acetate, 5—butan-1-ol, 6—propan-2-ol, 7—ethanol,
8—DMF, 9—DMSO, 10—methanol, 11—acetonitrile); Figure S7: Fluorescence lifetimes ( ) and fractional intensities
(%) measured for TSF, TS and TBrelative topH. Panel A—the main fluorescence lifetime component and panel
B—the main fluorescence lifetime component + the second component when present; Figure S8: DPPH
(200 M) absorption intensity decrease at max 519 nm in the presence of increasing concentration of tested
compounds after 30 min of reaction at 25 ◦C; Figure S9: Percentage of reduced DPPH
radicals under the influence
of increasing concentration of compounds tested after 30 minutes of reaction at 25 ◦C. The measurements were
taken at max 519 nm; Figure S10: Tandem mass spectrometry (MS/MS) of the thiadiazole derivatives studied:
(A) TB, (B) TS, and (C) TSF. The corresponding MS-chromatographic traces are given in inserts. The MS/MS
measurements were carried out using the collision energy of 30 eV; Table S1: Spectroscopic data. Maximum
absorbance, maximum fluorescence and Stokes shift in cm-1 for TB, TS, TSF; Table S2: Physical constants of
solvents. The average dipole molecular polarizability , dielectric constant , index of refraction n, functions,
F( ,n) and F( + n) + 2g(n) of the solvents; Table S3: Lifetimes ( ) and fractional intensities (f) measured for TSF,
•
radicals
µ
λ
•
λ
−
α
ε
ε
ε
τ
TS and TB depending for pH; Table S4: Thiadiazole derivatives MICs for 9 Candida species; Table S5: MIC curves
interpolations of thiadiazole derivatives against Candida species.
Author Contributions: Conceptualization: A.M. (Arkadiusz Matwijczuk); data curation: I.B., D.K., M.M., K.S.,
A.M. (Alicja Matwijczuk), B.M.-K., A.O., and and M.C.; formal analysis: D.K., M.M., K.R., K.S., A.M. (Alicja
Matwijczuk), B.M.-K., A.O., M.C., A.P., and A.M. (Arkadiusz Matwijczuk); funding acquisition: A.P. and A.M.
(Arkadiusz Matwijczuk); investigation: I.B., K.R., K.S., A.M. (Alicja Matwijczuk), B.M.-K., A.O., M.C., and
A.P.; methodology: D.K., M.M., K.R., A.M. (Alicja Matwijczuk), B.M.-K., A.O., and A.P.; project administration:
A.M. (Arkadiusz Matwijczuk); resources: D.K. and K.S.; software: B.M.-K., A.O., and M.C.; supervision: A.M.

Int. J. Mol. Sci. 2019, 20, 5494
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(Arkadiusz Matwijczuk); validation: I.B.; visualization: A.M. (Arkadiusz Matwijczuk); writing—original draft:
I.B., D.K., and A.M. (Arkadiusz Matwijczuk); writing—review and editing: M.M.
Funding: This research was financed by the Multi-Annual Program of Institute of Soil Science and Plant Cultivation,
State Research Institute, task 2.3: Assessment and support the implementation of integrated production processes
and technological progress in crop production (grain, forage and energy crops). This research was supported in
part also by PL-Grid Infrastructure. The research was carried out with the equipment purchased thanks to the
financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy
Operational Program (contract no. POIG.02.01.00-12-023/08). The author Arkadiusz Matwijczuk acknowledges
the Cost project CA 15126.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Rzeski, W.; Matysiak, J.; Kandefer-Szerszen´, M. Anticancer, neuroprotective activities and computational
studies of 2-amino-1,3,4-thiadiazole based compound. Bioorg. Med. Chem. 2007, 15, 3201–3207. [CrossRef]
[PubMed]
2.
3.
Kumar, D.; Kumar, N.M.; Chang, K.-H.; Shah, K. Synthesis and anticancer activity of 5-(3-indolyl)-
1,3,4-thiadiazoles. Eur. J. Med. Chem. 2010, 45, 4664–4668. [CrossRef] [PubMed]
Kadi, A.A.; El-Brollosy, N.R.; Al-Deeb, O.A.; Habib, E.E.; Ibrahim, T.M.; El-Emam, A.A. Synthesis,
antimicrobial, and anti-inflammatory activities of novel 2-(1-adamantyl)-5-substituted-1,3,4-oxadiazoles and
2-(1-adamantylamino)-5-substituted-1,3,4-thiadiazoles. Eur. J. Med. Chem. 2007, 42, 235–242. [CrossRef]
[PubMed]
4.
5.
6.
7.
8.
9.
Khan, I.; Ali, S.; Hameed, S.; Rama, N.H.; Hussain, M.T.; Wadood, A.; Uddin, R.; Ul-Haq, Z.; Khan, A.; Ali, S.;
et al. Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole
derivatives. Eur. J. Med. Chem. 2010, 45, 5200–5207. [CrossRef] [PubMed]
Matwijczuk, A.; Górecki, A.; Kamin´ski, D.; Mys´liwa-Kurdziel, B.; Fiedor, L.; Niewiadomy, A.; Karwasz, G.P.;
Gagos´, M. Influence of Solvent Polarizability on the Keto-Enol Equilibrium in 4-[5-(naphthalen-1-ylmethyl)-
1,3,4-thiadiazol-2-yl]benzene-1,3-diol. J. Fluoresc. 2015, 25, 1867–1874. [CrossRef]
Hoser, A.A.; Kamin´ski, D.M.; Skrzypek, A.; Matwijczuk, A.; Niewiadomy, A.; Gagos´, M.; Woz´niak, K. Interplay
of inter-and intramolecular interactions in crystal structures of 1,3,4-thiadiazole resorcinol derivatives.
Cryst. Growth Des. 2018, 18, 3851–3862. [CrossRef]
Kamin´ski, D.M.; Hoser, A.A.; Gagos´, M.; Matwijczuk, A.; Arczewska, M.; Niewiadomy, A.;
Woz´niak, K. Solvatomorphism of 2-(4-Fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole
Chloride. Cryst. Growth Des. 2010, 10, 3480–3488. [CrossRef]
Kamin´ski, D.M.; Matwijczuk, A.; Pociecha, D.; Górecka, E.; Niewiadomy, A.; Dmowska, M.; Gagos´, M. Effect
of 2-(4-fluorophenylamino)-5-(2, 4-dihydroxyphenyl)-1, 3, 4-thiadiazole on the molecular organisation and
structural properties of the DPPC lipid multibilayers. Biochi. Biophys. Acta. 2012, 1818, 2850–2859. [CrossRef]
Czernel, G.; Matwijczuk, A.; Karcz, D.; Górecki, A.; Niemczynowicz, A.; Szczes´, A.; Gładyszewski, G.;
Matwijczuk, A.; Gładyszewska, B.; Niewiadomy, A. Spectroscopic studies of dual fluorescence in
2-(4-fluorophenylamino)-5-(2, 4-dihydroxybenzeno)-1,3,4-thiadiazole: Effect of molecular aggregation
in a micellar system. Molecules 2018, 23, 2861. [CrossRef]
10. Zhou, P.; Hoffmann, M.R.; Han, K.; He, G. New insights into the dual fluorescence of methyl salicylate:
Effects of intermolecular hydrogen bonding and solvation. J. Phys. Chem. B 2014, 119, 2125–2131. [CrossRef]
11. Whitaker, J.E.; Haugland, R.P.; Prendergast, F.G. Spectral and photophysical studies of benzo [c] xanthene
dyes: Dual emission pH sensors. Anal. Biochem. 1991, 194, 330–344. [CrossRef]
12. Andersson, P.O.; Bachilo, S.M.; Chen, R.-L.; Gillbro, T. Solvent and temperature effects on dual fluorescence
in a series of carotenes. Energy gap dependence of the internal conversion rate. J. Phys. Chem. 1995, 99,
16199–16209. [CrossRef]
13. Ding, J.; Wang, Y.; Ma, M.; Zhang, Y.; Lu, S.; Jiang, Y.; Qi, C.; Luo, S.; Dong, G.; Wen, S.; et al. CT/fluorescence
dual-modal nanoemulsion platform for investigating atherosclerotic plaques. Biomaterials 2013, 34, 209–216.
[CrossRef] [PubMed]
14. Li, G.; Magana, D.; Dyer, R.B. Direct observation and control of ultrafast photoinduced twisted intramolecular
charge transfer (TICT) in triphenyl-methane dyes. J. Phys. Chem. B 2012, 116, 12590–12596. [CrossRef]

Int. J. Mol. Sci. 2019, 20, 5494
27 of 28
15. Sasaki, S.; Drummen, G.P.; Konishi, G.-I. Recent advances in twisted intramolecular charge transfer (TICT)
fluorescence and related phenomena in materials chemistry. J. Mater. Chem. C 2016, 4, 2731–2743. [CrossRef]
16. Padalkar, V.S.; Seki, S. Excited-state intramolecular proton-transfer (ESIPT)-inspired solid state emitters.
Chem. Soc. Rev. 2016, 45, 169–202. [CrossRef]
17. McDonald, L.; Wang, J.; Alexander, N.; Li, H.; Liu, T.; Pang, Y. Origin of water-induced fluorescence turn-on
from a Schiff base compound: AIE or H-bonding promoted ESIPT? J. Phys. Chem. B 2016, 120, 766–772.
[CrossRef]
18.
J
ó
wko, A.; Kowalczyk, J.; Wojciechowski, K. An XeCl* Excimer Fluorescence Study on the Pulse Radiolysis
of Xe–nCCl4 and Xe–SOCl2 Systems. J. Fluoresc. 2000, 10, 295. [CrossRef]
19. Brancato, G.; Signore, G.; Neyroz, P.; Polli, D.; Cerullo, G.; Abbandonato, G.; Nucara, L.; Barone, V.; Beltram, F.;
Bizzarri, R. Dual fluorescence through Kasha’s rule breaking: An unconventional Photomechanism for
intracellular probe design. J. Phys. Chem. B 2015, 119, 6144–6154. [CrossRef]
20. Yuan, Y.; Kwok, R.T.; Zhang, R.; Tang, B.Z.; Liu, B. Targeted theranostic prodrugs based on an
aggregation-induced emission (AIE) luminogen for real-time dual-drug tracking. Chem. Commun. 2014, 50,
11465–11468. [CrossRef]
21. Kwok, R.T.; Leung, C.W.; Lam, J.W.; Tang, B.Z. Biosensing by luminogens with aggregation-induced emission
characteristics. Chem. Soc. Rev. 2015, 44, 4228–4238. [CrossRef] [PubMed]
22. Starzak, K.; Matwijczuk, A.; Creaven, B.; Matwijczuk, A.; Wybraniec, S.; Karcz, D. Fluorescence
Quenching-Based Mechanism for Determination of Hypochlorite by Coumarin-Derived Sensors. Int. J.
Mol. Sci. 2019, 20, 281. [CrossRef]
23. Kasha, M.; Rawls, H.R.; El-Bayoumi, M.A. The exciton model in molecular spectroscopy. Pure Appl. Chem.
1965, 11, 371–392. [CrossRef]
24. Spano, F.C. The spectral signatures of Frenkel polarons in H-and J-aggregates. Acc. Chem. Res. 2009, 43,
429–439. [CrossRef] [PubMed]
25. Pasternack, R.F.; Collings, P.J. Resonance light scattering: a new technique for studying chromophore
aggregation. Science 1995, 269, 935–939. [CrossRef] [PubMed]
26. Matwijczuk, A.; Kaminski, D.; Górecki, A.; Ludwiczuk, A.; Niewiadomy, A.; Mackowski, S.; Gagos, M.
Spectroscopic Studies of Dual Fluorescence in 2-((4-Fluorophenyl) amino)-5-(2, 4-dihydroxybenzeno)-1,3,4-
thiadiazole. J. Phys. Chem. A 2015, 119, 10791–10805. [CrossRef] [PubMed]
27. Zhang, J.; Li, C.; Zhang, X.; Huo, S.; Jin, S.; An, F.-F.; Wang, X.; Xue, X.; Okeke, C.; Duan, G. In vivo
tumor-targeted dual-modal fluorescence/CT imaging using a nanoprobe co-loaded with an aggregation-
induced emission dye and gold nanoparticles. Biomaterials 2015, 42, 103–111. [CrossRef] [PubMed]
28. Patil, S.; Wari, M.; Panicker, C.Y.; Inamdar, S.; Wari, M. Determination of ground and excited state dipole
moments of dipolar laser dyes by solvatochromic shift method. Spectrochim. Acta Part. A Mol. Biomol. Spectrosc.
2014, 123, 117–126. [CrossRef]
29. Kawski, A. On the estimation of excited-state dipole moments from solvatochromic shifts of absorption and
fluorescence spectra. Z. Für Nat. A 2002, 57, 255–262. [CrossRef]
30. Prior, R.L.; Cao, G. In vivo total antioxidant capacity: Comparison of different analytical methods. Free Radic.
Biol. Med. 1999, 27, 1173–1181. [CrossRef]
31. Kedare, S.B.; Singh, R.P. Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol.
2011, 48, 412–422. [CrossRef] [PubMed]
32. Sharma, O.P.; Bhat, T.K. DPPH antioxidant assay revisited. Food Chem. 2009, 113, 1202–1205. [CrossRef]
33. Villaño, D.; Fernández-Pachón, M.; Moyá, M.L.; Troncoso, A.M.; Garcia-Parrilla, M.C. Radical scavenging
ability of polyphenolic compounds towards DPPH free radical. Talanta 2007, 71, 230–235. [CrossRef]
[PubMed]
34. Antinori, S.; Milazzo, L.; Sollima, S.; Galli, M.; Corbellino, M. Candidemia and invasive candidiasis in adults:
a narrative review. Eur. J. Intern. Med. 2016, 34, 21–28. [CrossRef] [PubMed]
35. Tragiannidis, A.; Tsoulas, C.; Groll, A.H. Invasive candidiasis and candidaemia in neonates and children:
Update on current guidelines. Mycoses 2015, 58, 10–21. [CrossRef] [PubMed]
36. Pfüller, R.; Gräser, Y.; Erhard, M.; Groenewald, M. A novel flucytosine-resistant yeast species, Candida
pseudoaaseri, causes disease in a cancer patient. J. Clin. Microbiol. 2011, 49, 4195–4202. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2019, 20, 5494
28 of 28
37. Parkash, J.; Robblee, J.H.; Agnew, J.; Gibbs, E.; Collings, P.; Pasternack, R.F.; de Paula, J.C. Depolarized
resonance light scattering by porphyrin and chlorophyll a aggregates. Biophys. J. 1998, 74, 2089–2099.
[CrossRef]
38. Reichardt, C.; Welton, T. Solvents and Solvent Effects in Organic Chemistry, 4th ed.; Wiley-VCH: Weinheim,
Germany, 2011.
39. Samundeeswari, S.; Kulkarni, M.; Yenagi, J.; Tonannavar, J. Dual fluorescence and solvatochromic study on
3-acyl coumarins. J. Fluoresc. 2017, 27, 1247–1255. [CrossRef]
40. Edward, J.T. Molecular volumes and the Stokes-Einstein equation. J. Chem. Educ. 1970, 47, 261. [CrossRef]
41. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.;
Mennucci, B.; Petersson, G.A.; et al. Gaussian 09 D.01 Revision A.1.; Gaussian: Wallingford, CT, USA, 2009.
42. Becke, A.D. Beckes 3 parameter functional combined with the non-local correlation LYP. J. Chem. Phys. 1993
98, 5648. [CrossRef]
,
43. Dunning, T.H., Jr. Gaussian basis sets for use in correlated molecular calculations. I. The atoms boron
through neon and hydrogen. J. Chem. Phys. 1989, 90, 1007–1023. [CrossRef]
44. Tomasi, J.; Mennucci, B.; Cammi, R. Quantum mechanical continuum solvation models. Chem. Rev. 2005
105, 2999–3094. [CrossRef] [PubMed]
,
45. Casida, M.E.; Jamorski, C.; Casida, K.C.; Salahub, D.R. Molecular excitation energies to high-lying bound
states from time-dependent density-functional response theory: Characterization and correction of the
time-dependent local density approximation ionization threshold. J. Chem. Phys. 1998, 108, 4439–4449.
[CrossRef]
46. Improta, R.; Scalmani, G.; Frisch, M.J.; Barone, V. Toward effective and reliable fluorescence energies in
solution by a new state specific polarizable continuum model time dependent density functional theory
approach. J. Chem. Phys. 2007, 127, 074504. [CrossRef]
47. Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Filamentous Fungi; Approved Standard, 2nd ed.; Clinical and Laboratory Standards Institute: Wayne,
PA, USA, 2008.
48. Morales, G.; Paredes, A.; Sierra, P.; Loyola, L. Antimicrobial activity of three Baccharis species used in the
traditional medicine of Northern Chile. Molecules 2008, 13, 790–794. [CrossRef]
49. Hoeflinger, J.L.; Hoeflinger, D.E.; Miller, M.J. A dynamic regression analysis tool for quantitative assessment
of bacterial growth written in Python. J. Microbiol. Methods 2017, 132, 83–85. [CrossRef]
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