Bichromophoric pyrazoline derivative with solvent-selective photoluminescence quenching

Article abstract of DOI:10.1016/j.molliq.2019.01.067

The quenching of fluorescence in the presence of chloromethanes, which is an unprecedented effect for pyrazolines, has been evidenced for the first time in the case of 1,3-diphenyl-5-{4-[(4-vinylbenzyl)oxy]phenyl}-4,5-dihydropyrazole. The detailed synthesis of this aryl trisubstituted pyrazoline that combines two chromophoric units in a non-conjugated manner is presented. The compound has been extensively characterized from a structural point of view, and its crystal structure has been determined by single crystal X-ray crystallography. The fluorescence study has evidenced the particular behavior of this pyrazoline derivative in solutions of chloromethanes, and the insight gained from the experimental data has been useful in elaborating a plausible fluorescence quenching mechanism. The investigated compound was modeled by Density Functional Theory (DFT) to point out the particularities of the electron transitions in gas phase as well as in the implicit solvents. Also, the HOMO-LUMO energy gap, mapped electrostatic potential, electronic density, dipole moment and polarizability have been reported for the pyrazoline derivative.

Full text of DOI:10.1016/j.molliq.2019.01.067

Accepted Manuscript
Bichromophoric pyrazoline derivative with solvent-selective
photoluminescence quenching
Andreea L. Chibac, Gheorghe Roman, Corneliu Cojocaru, Sergiu
Shova, Gabriela Sacarescu, Mihaela Simionescu, Liviu Sacarescu
PII:
S0167-7322(18)33190-8
DOI:
Reference:
https://doi.org/10.1016/j.molliq.2019.01.067
MOLLIQ 10298
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
21 June 2018
7 December 2018
12 January 2019
Please cite this article as: Andreea L. Chibac, Gheorghe Roman, Corneliu Cojocaru, Sergiu
Shova, Gabriela Sacarescu, Mihaela Simionescu, Liviu Sacarescu , Bichromophoric
pyrazoline derivative with solvent-selective photoluminescence quenching. Molliq (2019),
https://doi.org/10.1016/j.molliq.2019.01.067
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ACCEPTED MANUSCRIPT
Bichromophoric Pyrazoline Derivative with Solvent-Selective
Photoluminescence Quenching
Andreea L. Chibac, Gheorghe Roman, Corneliu Cojocaru, Sergiu Shova, Gabriela Sacarescu,
Mihaela Simionescu, Liviu Sacarescu*
Petru Poni Institute of Macromolecular Chemistry, 41 A Grigore Ghica Voda Alley, 700487,
Iasi, Romania
*e-mail: livius@icmpp.ro
Abstract
The quenching of fluorescence in the presence of chloromethanes, which is an unprecedented
effect for pyrazolines, has been evidenced for the first time in the case of 1,3-diphenyl-5-{4-
[(4-vinylbenzyl)oxy]phenyl}-4,5-dihydropyrazole. The detailed synthesis of this aryl
trisubstituted pyrazoline that combines two chromophoric units in a non-conjugated manner
is presented. The compound has been extensively characterized from a structural point of
view, and its crystal structure has been determined by single crystal X-ray crystallography.
The fluorescence study has evidenced the particular behavior of this pyrazoline derivative in
solutions of chloromethanes, and the insight gained from the experimental data has been
useful in elaborating a plausible fluorescence quenching mechanism. The investigated
compound was modeled by Density Functional Theory (DFT) to point out the particularities
of the electron transitions in gas phase as well as in the implicit solvents. Also, the HOMO-
LUMO energy gap, mapped electrostatic potential, electronic density, dipole moment and
polarizability have been reported for the pyrazoline derivative.
Keywords: Pyrazoline; Synthesis; Bichromophore; Fluorescence; Quenching; Molecular
modelling
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1. Introduction
An ever-growing research field is represented by sensors that could be used to
supervise various activities and processes, or even to monitor the health status of human
beings [1,2]. From this point of view, important efforts are concerned with light sensing and
response of materials or living organisms under light irradiation [3]. Fluorescence sensing is
already a well-known technique with particular applications in chemistry and biology [4].
This method is highly versatile, and its resolution down to molecular level is extremely useful
in molecular imaging [5]. To date, there are a huge number of organic molecules that were
reported as valuable fluorophores with different optical properties and sensing capabilities
[6]. An interesting class of compounds is represented by pyrazoles and their derivatives. In
the case of these compounds, several additional useful properties have been described beside
fluorescence. One of these applications refers to these compounds’ utility as active
compounds in the therapy of various medical conditions [7]. Thus, both optical and bio-active
properties could be combined in the same chemical structure to provide important
advantages, especially when the effect of the treatment should be monitored locally.
It is known that pyrazolines allow characteristic intramolecular charge transfer effects
that generate properties useful for various applications [8,9]. Particularly, 1,3,5-triaryl-2-
pyrazoline derivatives that possess fluorescence in the blue region of the spectrum have been
employed also as hole transporting materials, organic electroluminescent devices, fluorescent
probes for chemosensors and fluorescent switches [10]. In all of these examples, pyrazolines
have been reported as ligands in complex metal-organic systems in which the nature of the
cation is crucial to the materialization of the desired property [11,12].
This paper describes a pyrazoline having specific properties, distinctive than those
reported for other members of the class. Hence, 1,3-Diphenyl-5-{4-[(4-
vinylbenzyl)oxy]phenyl}-4,5-dihydropyrazole has a bichromophoric structure that enables a
selective blue emission as a function of the solvent employed. Thus, fluorescence
measurement experiments conducted for this pyrazoline in chloromethanes as solvents have
resulted in quenching, whereas the same pyrazoline exhibits a strong emission and a quantic
yield higher than 75% in various other polar and non-polar solvents. This selectivity could be
exploited for the development of detection sensors that operate through fluorescence
quenching, and are effective for the identification, monitoring and eventually treatment of
medical conditions for which the local modification of chemical composition of the tissue is
indicative.
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2. Experimental
2.1. Materials, methods and instrumentation
All the chemical reagents and solvents were obtained from Sigma–Aldrich
(Schnelldorf, Germany) and were used without prior purification. The optical studies were
performed using UV-grade solvents. Melting points were taken on a MEL-TEMP capillary
melting point apparatus and are uncorrected. Elemental analysis was conducted on a
1
13
PerkinElmer 2400 Series II CHNS/O system. H and C NMR spectra were recorded on a
Bruker Avance 400-MHz spectrometer. The signals owing to residual protons in the
1
deuterated solvents were used as internal standards for the H NMR spectra. The chemical
shifts for the carbon atoms are given relative to deuteriochloroform (δ = 77.16 ppm) and
DMSO-d6 (δ = 39.52 ppm). Fourier Transform-Infrared (FT-IR) spectra were taken on a FT-
IR Bruker Vertex 70 instrument in transmission mode, using KBr pellets. The UV-Vis
absorption spectrum was recorded on a SPECORD 210 Plus Analytik Jena
spectrophotometer. Fluorescence spectra were collected on a Perkin-Elmer LS 55
spectrofluorometer. Single crystal X-ray measurements were carried out using
Oxford‒Diffraction XCALIBUR E CCD equipment set up with graphite‒monochromated
Mo‒Kα radiation.
2.2. Chemistry
2.2.1. Synthesis of 4-[(4-vinylbenzyl)oxy]benzaldehyde 1
To a solution of KOH (658 mg, 10 mmol, 85% purity) in ethanol 96% (10 mL), 4-
hydroxybenzaldehyde (1.22 g, 10 mmol) was added, and then the mixture was stirred at room
temperature for 5 min. 4-Vinylbenzyl chloride (1.694 mg, 10 mmol, 90% purity) was then
added, and then the reaction mixture was heated at reflux temperature for 2 h. Aqueous 5%
KOH (2 mL) was then gradually added to the hot mixture, followed by water (30 mL), and
the mixture was further stirred for 30 min. The resulting solid was filtered, air-dried, and
1
recrystallized to give colorless crystals (1.71 g, 72%), mp 76–77 °C (methanol). H NMR
(CDCl3, 400 MHz): δ 5.14 (s, 2H), 5.28 (d, J = 10.4 Hz, 1H), 5.78 (d, J = 17.6 Hz, 1H), 6.73
(dd, J = 10.4 and 17.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.45 (d, J =
13
8.0 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 9.89 (s, 1H). C NMR (CDCl3, 100 MHz): δ 70.2,
114.6, 115.3, 126.7, 127.8, 130.3, 132.1, 135.5, 136.4, 137.8, 163.8, 190.9. Anal. Calcd. for
C16H14O2: C 80.65; H 5.92. Found: C 80.41; H 5.73.
2.2.2. Synthesis of (E)-1-phenyl-3-{4-[(4-vinylbenzyl)oxy]phenyl}prop-2-en-1-one 2
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4-[(4-Vinylbenzyl)oxy]benzaldehyde 1 (1428 mg, 6 mmol) was dissolved in warm
methanol (60 mL) with efficient stirring. The solution was cooled to room temperature, then
acetophenone (720 mg, 6 mmol) and 10% NaOH (1 mL) were sequentially added. The
mixture was stirred at room temperature overnight, then the solid that had separated was
filtered, washed sequentially with 2-propanol (2× 10 mL) and water (2× 10 mL), air-dried,
and recrystallized to afford yellowish leaflets (1080 mg, 53%), mp 104–105 °C (methanol).
1H NMR (CDCl3, 400 MHz): δ 5.10 (s, 2H), 5.28 (d, J = 10.4 Hz, 1H), 5.78 (d, J = 17.6 Hz,
1H), 6.73 (dd, J = 10.4 and 17.6 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 7.36–7.47 (m, 5H), 7.47–
13
7.55 (m, 2H), 7.55–7.64 (m, 3H) 7.79 (d, J = 15.6 Hz, 1H), 8.02 (d, J = 7.2 Hz, 2H). C
NMR (CDCl3, 100 MHz): δ 70.0, 114.4, 115.4, 120.0, 126.6, 127.8, 128.0, 128.5, 128.7,
130.4, 132.7, 136.0, 136.5, 137.7, 138.6, 144.7, 160.9, 190.7. Anal. Calcd. for C24H20O2: C
84.68; H 5.92. Found: C 84.40; H 6.15.
2.2.3. Synthesis of 1,3-diphenyl-5-{4-[(4-vinylbenzyl)oxy]phenyl}-4,5-dihydro-1H-pyrazole 3
To a solution of (E)-1-phenyl-3-(4-([4-vinylbenzyl]oxy)phenyl)prop-2-en-1-one 2
(1020 mg, 3 mmol) in glacial acetic acid (20 mL), phenylhydrazine (432 mg, 4 mmol) is
added. The solution is stirred at room temperature overnight, then the solid that separated was
filtered, washed with ethanol (2× 10 mL), air-dried and recrystallized twice from ethyl
acetate–2-propanol (1:3, v/v) to afford off-white crystals (465 mg, 36%), mp 139–140 °C.
FTIR (KBr, max [cm-1]): 685s, 747s, 824s, 987m, 1052m, 1129s, 1172m, 1216m, 1242vs,
1329m, 1390s, 1496vs, 1587vs, 1656w, 2835w, 2852w, 2885w, 2911w, 2921w, 2976w,
3027m, 3035m, 3052m, 3067w, 3129w. 1H NMR (DMSO-d6, 400 MHz): δ 3.07 (dd, J = 6.0
and 13.2 Hz, 1H), 3.86 (dd, J = 12.0 and 13.2 Hz, 1H), 5.03 (s, 2H), 5.25 (d, J = 11.2 Hz,
1H), 5.41 (dd, J = 6.0 and 12.0 Hz, 1H), 5.83 (d, J = 18.0 Hz, 1H), 6.66–6.79 (m, 2H), 6.96
(d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 7.10–7.18 (m, 2H), 7.21 (d, J = 8.8 Hz, 2H),
13
7.32–7.51 (m, 7H), 7.74 (d, J = 7.6 Hz, 2H), C NMR (DMSO-d6, 100 MHz): δ 43.0, 62.6,
68.9, 113.0, 114.5, 115.2, 118.6, 125.7, 126.2, 127.1, 128.0, 128.7, 128.9, 132.4, 134.7,
136.3, 136.7, 144.3, 147.2, 157.6. Anal. Calcd. for C30H26N2O: C 83.69; H 6.09; N 6.51.
Found: C 83.44; H 6.17; N 6.33.
2.3. Single-crystal X-ray diffraction study
A single-crystal of pyrazoline 3, which has been obtained through the slow diffusion of
methanol into a solution of the compound in dichloromethane, was used for the
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crystallographic data collection. The unit cell estimation and data integration were done using
the CrysAlis package of Oxford Diffraction [13]. The molecular structure was solved by
direct methods using Olex2 program [14] and refined by full-matrix least-squares on F² with
SHELXL-97 [15]. The molecular-structure plot was rendering by Olex2 software.
2.4. UV-Vis and fluorescence measurements
The absorption spectrum of compound 3 was recorded either in toluene or chloroform
solutions at room temperature, using 10 mm quartz cells. The absorption maximum of
pyrazoline 3 was used as excitation wavelength (λ = 365 nm) for all fluorescence spectra. The
excitation slits were set at 15 and 0 nm for the measurements in toluene and DMSO solutions,
respectively. Due to the weak fluorescence emission the measurements in chloroform and
carbon tetrachloride, were done by amplifying the signal with the excitation slits set at 5 and
8 nm. The concentration of compound 3 in each solvent was 5×10-3 wt%. In toluene and
chloroform, the fluorescence spectra were recorded at different concentrations in the range of
10-4–5×10-2 wt%. For the quenching experiment using the toluene solution of pyrazoline 3,
the quencher concentration of chloroform was varied in the range 0.0 to 23.33 wt%.
2.5. Molecular modeling
All computations were done using the Gaussian09 [16] software package on a Dell
server with 24 computing cores. Molecular modeling results were analyzed in GaussView 5
graphical-interface program [17] running on a Dell Precision workstation T7910. Density
functional theory (DFT) methods (B3LYP and CAM-B3LYP) were employed for the
molecular modeling using split-valence basis sets. The geometry optimization of the
investigated molecule was done by minimizing the energy with respect to all geometrical
parameters without imposing any symmetry constrains. The input structure for optimization
was taken from the CIF-file derived from X-ray single-crystal measurements. Subsequently,
the geometry optimization assisted by frequency calculation was performed on a single
molecule in gas phase. All optimized geometries resulted out of different basis sets unveiled
C1 point group and no imaginary frequencies, thereby confirming the minimum-energy
structures. The electronic absorption spectra of pyrazoline 3 were predicted by the time-
dependent density functional theory (TD-DFT). In addition, the YASARA program [18] was
employed for conformational analysis and explicit solute-solvent interaction simulation.
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3. Results and Discussion
A fluorescent pyrazoline having a vinyl moiety has been synthesized with the
intention to develop new fluorescence sensors (Scheme 1). The vinyl moiety at position 5 of
the designed pyrazoline has been put in place using commercially available 4-vinylbenzyl
chloride, which was reacted with 4-hydroxybenzaldehyde under the conditions of Williamson
ether synthesis (Scheme 1). Next, Claisen-Schmidt condensation of the resulting vinyl-
containing aldehyde 1 with acetophenone was conducted in a large volume of methanol, in
the presence of catalytic amounts of NaOH, to afford the intermediate chalcone 2, which led
to the desired pyrazoline 3 upon condensation with phenylhydrazine in acetic acid in the final
step of the reaction sequence. A similar synthetic strategy has been used by Qi et al. for the
preparation of the same compound [19].
CHO
OH
CH2Cl
b
a
OHC
O
1
+
CH CH2
O
c
N
N
O
O
2
3
Scheme 1. Synthesis of fluorescent pyrazoline 3. Reactants and conditions: (a) ethanol 96%,
KOH, reflux, 2 h; (b) acetophenone, methanol, NaOH, room temperature, 18 h; (c)
phenylhydrazine, glacial acetic acid, room temperature, 18 h.
Aldehyde 1 is a known compound, whose synthesis from the same starting materials
has been reported using several approaches, and the NMR characteristics of this compound,
which have been only recently fully described [19-23], are in agreement with those
previously reported. To the best of our knowledge, compounds 2 and 3 have only been
mentioned once in the literature [19], although pyrazoline 3 appears to have been previously
employed also as a fluorescent model drug to investigate the internalization of aggregates of
an amphiphilic copolymer by cells [24]. Careful duplication of the procedure previously
reported [19] has led in our hands to the isolation of a mixture of unreacted starting material
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and reaction product. Nevertheless, the extension of the reaction time up to 18 h allowed the
separation of a crude material consisting mostly of the desired compound, from which
pyrazoline 3 was obtained in pure form only after repeated recrystallizations, which occurred
with significant loss of material and lowered the yield.
The structure of the target compound 3 has been established by NMR spectroscopy.
The proton spectrum of pyrazoline 3 has been previously described [19], but no comments on
its characteristics have been provided. The structure of pyrazoline 3 was confirmed through
the identification of the three doublets of doublets centered at 3.07, 3.86 and 5.41 ppm,
corresponding to the protons at C-4 and C-5 of the pyrazoline ring. Formation of pyrazoline 3
has been also substantiated through the presence of two peaks in the aliphatic region of the
13C NMR spectrum of this compound, corresponding to C-4 (43.0 ppm) and C-5 (63.6 ppm).
The synthesized pyrazoline 3 afforded single crystals upon slow diffusion of methanol
into its solution in dichloromethane. They were further analyzed using X-ray diffraction to
obtain the crystallographic data and refinement details presented in ESI Table S1.
The molecular structure observed for pyrazoline 3 is shown in Figure 1. The
supramolecular packing structure was formed through CH…-aromatic interactions pointed
out by hydrogen-to-centroid distance ranging from 2.724 Å to 3.025 Å (ESI, Fig. S1).
Figure 1. Molecular structure of pyrazoline 3 resulted from single crystal X-ray structure
analysis (Olex2 view), thermal ellipsoids drawn at 50% probability level.
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UV-Vis and fluorescence (FL) spectra of pyrazoline 3 in either chloroform or toluene
are presented in Figure 2. Any attempt to interpret the UV-Vis spectrum of pyrazoline 3 has
to consider the particular structure of this compound, which consists of two chromophores
represented by the two separate, uncoupled conjugated electronic systems [20,26]. One of the
chromophores (C1) is represented by the phenyl groups connected through the hydrazone
bridge within the pyrazoline core, and the other (C2) is the vinylbenzyloxyphenyl substituent
at position 5 of the pyrazoline ring. Consequently, two different electron transitions
correlated with these conjugated systems should be present in the UV-Vis spectrum of
pyrazoline 3 (Fig. 2a). These electronic transitions can be best observed when the UV
absorption spectrum of pyrazoline 3 is recorded in chloroform. One of them is located at 260
nm, and it was assigned to the -* electronic transitions in the C2 system. The other UV
absorption peak is noticeable around 362 nm, and it was associated with the -*electron
transitions in the C1 chromophore. Due to the solvent cut-off, the UV spectrum of pyrazoline
3 in toluene displayed only the C1 peak, which is located also at 362 nm. Moreover, the
position, the intensity, and even the shape of the UV absorption band assigned to the
pyrazoline core (C1) are not affected by the nature of the solvent, which suggests that the C1
ground state is not perturbed in any solvent, whether the solvent is either mostly non-polar
(toluene) or slightly polar (chloroform).
Figure 2. (a) Absorption and (b) emission (λex = 365 nm) spectra of pyrazoline 3 (solution
concentration is 5×10-4 wt%). In order to have a measurable fluorescence intensity, the
emission spectra in CHCl3 and CCl4 were recorded in special conditions.
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The fluorescence spectrum of pyrazoline 3 (Fig. 2b) taken in toluene differs
significantly from the one recorded in chloroform. Thus, at λex = 365 nm, the FL spectrum in
both solvents showed only a single emission maximum at 450 nm. Surprisingly, the emission
intensities were very different for each solution: the emission intensity was high in toluene
(quantum yield of 75%) and almost negligible in chloroform (quantum yield lower than
15%). This result pointed out that chloroform had an important fluorescence quenching effect
on pyrazoline 3.
With the view to explain the almost complete quenching of the emission intensity of
pyrazoline 3 in chloroform, which represents an unprecedented behavior for pyrazolines, a
series of additional experiments have been designed and performed. In order to examine the
emission spectra of pyrazoline 3 in solvents having diverse structures and physical properties,
several solvents have been selected. First, the FL spectrum of compound 3 has been recorded
in dimethylsulfoxide (DMSO), a highly polar solvent, and compared to the one recorded in
toluene, which is a non-polar solvent. The results have shown that the polarity of the solvent
has little influence of the emission of pyrazoline 3, as only slight bathochromic and
hyperchromic effects could be noticed for the emission spectrum recorded in DMSO (Fig. 3.).
A significant bathochromic effect is usually induced by a highly polar solvent and is a
consequence of the photoexcitation process leading to a reorientation of the solvent
molecules in the proximity of the fluorophore. Subsequently, part of the energy of the excited
molecules of the fluorophore is transferred to the solvent, resulting in a batochromic effect.
Owing to the presence of two chromophores in the structure of pyrazoline 3, the magnitude of
the aforementioned effect in DMSO is small and the Stokes shift is only 25 nm. The
explanation is that processes involving both chromophores such as a significant geometrical
relaxation of the molecule in the excited state and the internal charge transfer require a
substantial part of the transferred energy.
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Figure 3. Fluorescence spectra of pyrazoline 3 in toluene (black line) and DMSO (red line);
(λex = 365 nm).
Next, the emission spectrum of pyrazoline 3 was recorded in carbon tetrachloride, a
solvent which is non-polar, but is structurally related to chloroform (Fig. 2b). The use of
carbon tetrachloride in these experiments led again to an almost complete FL quenching, in a
manner similar to that observed for the solutions of pyrazoline 3 in chloroform. Moreover,
the fluorescence quenching was so intense in chloroform and carbon tetrachloride that, in
order to evidence the small emission of pyrazoline 3, these experiments have to be performed
under conditions that allowed the amplification of the signal as described in the experimental
part. These results strongly suggest that chloromethanes quench the fluorescence of
pyrazoline 3. Further research concerning this phenomenon has been made by studying the
FL quenching effect of chloroform in pyrazoline 3 solutions in toluene. Thus, addition of
increasing amounts of chloroform to a toluene solution of pyrazoline 3 resulted in a decrease
of FL intensity, as shown in Figure 4. Although the same effect has been reported for
compounds having different structures [27-29], it has been hitherto unknown for 1,3,5-
triarylpyrazolines.
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Figure 4. Fluorescence quenching effect of chloroform on the solution of pyrazoline 3 in
toluene (λex = 365 nm).
Fluorescence quenching of a chromophore in the presence of halogenated alkanes is
normally due to the formation of non-emissive exciplexes [27,30,31]. Generally, fluorescence
quenching in such systems could be based either on a static mechanism, or a dynamic
mechanism, or both. The static mechanism stipulates the formation of a charge transfer
complex between the molecules of fluorophore in the excited state and those of the
halogenated alkane. In contrast to the dynamic mechanism, the processes that are
characterized by a static mechanism do not involve the diffusion of the molecules of the
halogenated alkane towards the fluorophore. Valuable insight on the actual mechanism
through which the fluorescence quenching of pyrazoline 3 by addition of chloroform in
toluene solutions takes place can be gained from the chart in Figure 5 by using the Stern-
Volmer equation. This chart has obtained by plotting the I0/I ratio as a function of the
concentration of the quencher [Q].
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Figure 5. Fitting of the Stern-Volmer curve describing the nonlinear behavior of the
fluorescence of pyrazoline 3 upon quenching with chloroform; I0 represents the fluorescence
intensity in the absence of the quencher, and I is the fluorescence intensity in the presence of
the quencher’s concentration [Q].
The dependence between I0/I and [Q] could be linear, and, in this case, the quenching
takes place through a dynamic, collisional mechanism, which is largely controlled by
diffusion. In our case, however, the chart obtained for the quenching of the fluorescence of
pyrazoline 3 by chloroform exhibits a strong positive deviation (Fig. 5). The profile of the
curve is typical for the situation in which the quenching occurs through a complex
combination of both mechanisms. Thus, as some of the molecules in the excited state undergo
a dynamic quenching through collisions, the rest of the fluorophore’s molecules is
deactivated instantaneously, as soon as they were excited. The static component of the
quenching mechanism is the result of the presence of quencher molecules being randomly
situated in the vicinity of the fluorophore’s molecule as excitation occurs. One of the methods
used to determine the contribution of the two mechanisms to the fluorescence quenching
process consists in the calculation of the constants for dynamic and static quenching using the
appropriate form of the Stern-Volmer equation. In our particular case, a modified Stern-
Volmer equation [32] has been used, as follows:
I0/I = 1+ Ksv[Q]exp(V[Q]),
where Ksv and V are the dynamic constant and the static constant, respectively.
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The fitting of the experimental data onto the curve that has been generated using the
above equation shows a perfect correlation between the theoretical model and experimental
data (residual sum of squares, RSS = 3.4×10-4), and, therefore, the calculation of the two
constants using the modified Stern-Volmer equation is viable. The values obtained for these
two constants (Ksv = 1.3×10-2 M-1; V = 2.033 M-1) are indicative of a predominantly static
quenching mechanism. Considering the excellent correlation between experimental data and
the theoretical model, as well as the observation that the shift of the fluorescence intensity in
the presence and absence of the quencher is insignificant, a plausible model for the
fluorescence quenching process is the sphere-of-static action. This model presumes the
existence of a defined volume inside which the whole quenching process takes place [33,34],
and, according to this model, the size of the sphere can be estimated using the following
equation:
v = 1000 V/N = 4r3/3,
where N is Avogadro’s number (6.022×1023/mole), and r is the radius of the sphere
surrounding the fluorophore’s molecule during fluorescence quenching.
In our case, the volume of the sphere can be calculated using the value of the static
quenching constant (as determined with the modified Stern-Volmer equation):
v = 3×1000 V/N= 498.1×10-23 = 4981 Å3, and
r = [3 x 4981/4r = 10.59 Å
This value corresponds to a structure in which the molecules of chloroform are
positioned preferentially on both sides of the plane that includes the aromatic ring of
chromophore C2. The layout has been also confirmed by molecular modeling, as shown
below.
Although the sphere-of-static action model fits perfectly the experimental data, this
model cannot theoretically exclude the possibility of the presence of elements belonging to
the dark complex model. This latter model assumes the formation of a particular type of
complex between the fluorophore’s molecules in the ground state and the quencher’s
molecules prior to excitation [35]. This specific type of complex does not imply a physical
contact between the molecules involved in its formation, but rather a positioning of the
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quencher’s molecules close to the fluorophore’s molecule as the result of subtle interactions
between the two constituents of the complex. In this case, the unbound fluorophore is the
source of fluorescence, and, according to this model, V is rather an association constant. The
contribution of the dark complex model to the fluorescence quenching mechanism of
pyrazoline 3 is justified by the existence of a minor tendency for the fluorescence intensity to
shift towards lower wavelength numbers with the significant increase of the quencher’s
concentration. Therefore, although the noticeable effect is extremely weak, it may be
potentially explained by a static quenching due to the presence of a slight association between
the fluorophore and the quencher similar to that in dark complexes.
The essential characteristics of the particular electronic structure that was created by
joining in a non-conjugated manner the two chromophores C1 and C2 within the molecule of
pyrazoline 3 can be detailed with the help of the molecular model generated for this
compound by quantum chemical methods (details in ESI - Pyrazoline 3 molecular model).
The generation of a molecular model capable to reflect adequately the structure of the
pyrazoline 3 has relied greatly on the information gleaned from the single crystal X-ray
experiment.
Details regarding geometry optimization and full description of the method are
presented in ESI (Table S2 and Fig. S2). Thus, the DFT methods B3LYP and CAM-B3LYP
were employed for geometry optimization of pyrazoline 3 (Fig. 6). A comparison between
the X-ray measured and predicted geometries indicated the best method in terms of root-
mean-square-deviation (RMSD).
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Figure 6. (a) Optimal geometry computed by DFT method at B3LYP/6-31G** level in
ground state. (b) Superposition of molecular structures of pyrazoline 3, X-ray structure (blue)
and theoretical structures (red) computed by B3LYP/6-31G** (RMSD=0.5903 Å).
In the next step, the molecule geometry was calculated using the methodology for
explicit solvents [36]. The geometries were validated by comparison of the computed
electronic spectra with the experimental ones [37,38]. Thus, the predicted absorption maxima
of 363.9 nm for chloroform and 356.2 nm for toluene were in good agreement with the
experimental ones.
Then, the theoretical UV-Vis spectra were simulated in chloroform and toluene as
implicit solvents using the polarizable continuum model (PCM) with the integral equation
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formalism (IEFPCM) [39]. The best results were obtained when the theoretical spectra were
predicted using CAM-B3LYP/6-31G** (Fig. 7).
Figure 7. The electronic absorption spectrum computed by TD-DFT method at CAM-
B3LYP/6-31G** level using IEFPCM model for implicit solvents: (a) chloroform and (b)
toluene.
The molecular orbital analysis showed that the main contribution (52-53%) to the 242
nm transition represented the HOMO-2  LUMO+1 electronic configuration. The lobes of
these molecular orbitals (HOMO-2 and LUMO+1) were mainly spread onto the
vinylbenzyloxy moiety C2 (Fig. 8).
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Figure 8. Molecular orbitals involved in the transition at the excitation energy of 242 nm
along with assignments and percentage contributions; computation done at CAM-B3LYP/6-
31G** level of theory.
Under the influence of explicit solvents, the lobes of HOMO are also distributed
throughout the C1 moiety in both solvents (Fig. 9). In toluene, most of the LUMO pattern is
also located on C1, with no conjugation to the second C2 chromophore (Fig. 9b). On the
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other hand, in the case of chloroform, the lobes of LUMO are delocalized on both C1 and the
adjacent chloroform molecule (Fig. 9a).
Figure 9. Patterns of frontier molecular orbitals (HOMO-LUMO) for pyrazoline 3 influenced
by the presence of explicit solvent molecules (nearby SAS): (a) the effect of chloroform
solvent and (b) the effect of toluene solvent.
All these calculations indicate that the presence of vinylbenzyloxy moiety C2 in
pyrazoline 3 generate structural particularities that lead to optical and electronic properties
strongly influenced by the presence of neighbouring chloroform molecules. This observation
sustains the hypothesis and mechanism concerning the fluorescence quenching by
chloromethanes.
Conclusions
A fluorescent pyrazoline having a vinyl moiety has been synthesized with the intention to
develop new fluorescence sensors. For this purpose 4-vinylbenzyl chloride was reacted with
4-hydroxybenzaldehyde under the conditions of Williamson ether synthesis. Next, Claisen-
Schmidt condensation of the resulting vinyl-containing aldehyde with acetophenone afford
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the intermediate chalcone, which led to the desired pyrazoline 3 upon condensation with
phenylhydrazine in acetic acid. The bichromophoric nature of this compound and the
presence of the 4-vinylbenzyl)oxy]phenyl moiety are structural particularities that lead to
specific fluorescence properties. Thus, the study of the optical properties in various solvents
showed that the fluorescence of pyrazoline 3 is selectively quenched by chloroalkanes. The
fluorescence quenching mechanism was analyzed considering chloroform as quencher. Stern-
Volmer equation plot indicates a complex quenching mechanism with a strong static
component. Molecular modeling of pyrazoline 3 was used to give details related to the cause
of the fluorescence quenching by chloromethanes. Advanced computational techniques
evidenced that, a non-radiative relaxation of the excited pyrazoline 3 is possible due to the
formation of a specific HOMO-LUMO molecular orbital configuration that involves the
chloromethane surrounding molecules. The fluorescence and bioactive properties of
pyrazoline 3 have a high potential utility in development of sensors for detection and
treatment of specific medical illness.
Acknowledgement
The computational chemistry section of the manuscript is part of a project that has received
funding from the European Union's Horizon 2020 research and innovation program under the
grant agreement No 667387 WIDESPREAD 2-2014 SupraChem Lab.
A.L. Chibac is thankful for the financial support offered by CNCSIS-UEFISCDI, project
number PN-III-P1-1.1-TE-2016-1390 (No. 34 ⁄ 2018).
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Graphical abstract
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Highlights
 fluorescence quenching of pyrazoline derivative by chloroalkanes.
 synthesis of aryl trisubstituted pyrazoline with two non-conjugated chromophoric
units.
 structural and optical particularities
 the fluorescence quenching mechanism.
 molecular modeling of pyrazoline by Density Functional Theory in implicit solvents.
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