Synthesis of new styrylquinoline cellular dyes, fluorescent properties, cellular localization and cytotoxic behavior

Article abstract of DOI:10.1371/journal.pone.0131210

New styrylquinoline derivatives with their photophysical constants are described. The synthesis was achieved via Sonogashira coupling using the newly developed heterogeneous nano-Pd/Cu catalyst system, which provides an efficient synthesis of high purity

Full text of DOI:10.1371/journal.pone.0131210

RESEARCH ARTICLE
Synthesis of New Styrylquinoline Cellular
Dyes, Fluorescent Properties, Cellular
Localization and Cytotoxic Behavior
Marzena Rams-Baron1,2☯, Mateusz Dulski2,4, Anna Mrozek-Wilczkiewicz1,2
,
Mateusz Korzec3☯, Wioleta Cieslik3, Ewelina Spaczyńska3, Piotr Bartczak3,
Alicja Ratuszna1,2, Jaroslaw Polanski3, Robert Musiol3*
1 A. Chelkowski Institute of Physics, University of Silesia, Uniwersytecka 4, Katowice, 40–007, Poland,
2 Silesian Center for Education and Interdisciplinary Research, 75 Pulku Piechoty 1A, Chorzow, 41–500,
Poland, 3 Institute of Chemistry, University of Silesia, Szkolna 9, Katowice, 40–006, Poland, 4 Institute of
Material Sciences, University of Silesia, 75 Pulku Piechoty 1A, Chorzow, 41–500, Poland
☯ These authors contributed equally to this work.
* robert.musiol@us.edu.pl
Abstract
OPEN ACCESS
New styrylquinoline derivatives with their photophysical constants are described. The syn-
thesis was achieved via Sonogashira coupling using the newly developed heterogeneous
nano-Pd/Cu catalyst system, which provides an efficient synthesis of high purity products.
The compounds were tested in preliminary fluorescent microscopy studies to in order to
identify their preferable cellular localization, which appeared to be in the lipid cellular organ-
elles. The spectroscopic properties of the compounds were measured and theoretical TD-
DFT calculations were performed. A biological analysis of the quinolines that were tested
consisted of cytotoxicity assays against normal human fibroblasts and colon adenocarci-
noma cells. All of the compounds that were studied appeared to be safe and indifferent to
cells in a high concentration range. The presented results suggest that the quinoline com-
pounds that were investigated in this study may be valuable structures for development as
fluorescent dyes that could have biological applications.
Citation: Rams-Baron M, Dulski M, Mrozek-
Wilczkiewicz A, Korzec M, Cieslik W, Spaczyńska E,
et al. (2015) Synthesis of New Styrylquinoline Cellular
Dyes, Fluorescent Properties, Cellular Localization
and Cytotoxic Behavior. PLoS ONE 10(6): e0131210.
doi:10.1371/journal.pone.0131210
Editor: A Ganesan, University of East Anglia,
UNITED KINGDOM
Received: February 7, 2015
Accepted: May 29, 2015
Published: June 26, 2015
Copyright: © 2015 Rams-Baron et al. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Introduction
Data Availability Statement: All relevant data are
within the paper.
Quinoline-based compounds are of special interest to organic and medical chemists due to
their variety of applications, which are not limited only to pharmaceutical uses. Such com-
pounds, which have extended delocalized π-electron systems, may exhibit exciting optical
properties that may offer opportunities for potential applications such as optical materials or
luminescent probes. Styrylquinolines are an excellent example of such applicable and biologi-
cally relevant materials. They are known antiviral agents that are a styrylquinoline inhibitors of
HIV integrase [1], antifungal [2] and anticancer agents [3]. Particular attention has been paid
to this class of compounds because of their electroluminescence and photochromic properties,
which could permit their potential utility in optical devices or as biosensors [4,5]. Many reports
Funding: This study was supported by NCN—
National center for science grants 2014/13/D/NZ7/
00322 (AMW), 2012/07/N/NZ7/02110 (WC), 2013/09/
B/NZ700423 (RM) and DEC-2012/07/N/ST5/02221
(MD); National Research and Development Center
(NCBiR) Grant ORGANOMET no: PBS2/A5/40/2014.
This research was supported in part by PL-Grid
Infrastructure.
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Competing Interests: The authors have declared
that no competing interests exist.
have pointed out the feasible use of molecules such as fluorophores that could be suitable for
staining intracellular components such as nucleic acids or proteins [6,7]. Recently, fluorescent
styrylquinoline was reported as an application as an amyloid imaging sensor, which could be
useful in diagnosing Alzheimer’s disease [8].
In our group, quinoline derivatives are being broadly investigated for their chemistry [9,10]
and possible applications [11–13]. These researches have resulted in some highly active anti-
cancer [14,15] and antifungal [16] agents. In the present study, we describe the structural, elec-
tronic and optical properties of novel styrylquinolines and their simple analogs. The concept of
the design of quinoline dyes is depicted in Fig 1. It combines the alkyne analogs of 1a-b and
styrylquinolines with an elongated lipophilic chain 3a-d.
Such structural features are necessary for good fluorescent properties. On the other hand,
the compounds do not have the functional groups that are required for their biological activity,
e.g., the hydroxyl group at the C-8 position in a quinoline ring. This may suggest their low
cytotoxicity and relative indifference to biological systems. With this in mind, we applied
steady state UV-VIS absorption and emission spectroscopy in order to characterize their
photophysical behavior. We applied ab initio molecular computing using the density func-
tional theory (DFT) and time-dependent DFT (TD-DFT) calculations to gain insight into the
electronic structure of the molecules that were being considered and in order to fully under-
stand the experimental data. The impact of the structural features on the emission efficiency of
styrylquinoline dyes is particularly important in terms of the development of novel fluoro-
phores that might be suitable for various labeling applications.
Materials and Methods
A. General experimental method
All of the reagents were purchased from Aldrich. A Kieselgel 60, 0.040–0.063 mm (Merck,
Darmstadt, Germany) was used for column chromatography. The TLC experiments were per-
formed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Germany). The plates
were illuminated under UV (254 nm). The melting points were determined on an Optimelt
MPA-100 apparatus (SRS, Stanford CA). The purity of the final compounds was checked using
HPLC. Detection wavelengths of 210 and 250 nm were chosen for detection. The purity of indi-
vidual compounds was determined from the area peaks in the chromatogram of the sample
solution in order to ensure >95% purity. UV spectra (λ, nm) were determined on a Waters
2996 Photodiode Array Detector (Waters Corp., Milford, MA, U.S.A.) in a methanolic solution
(ca. 6×10-4mol) and log ε was calculated for the absolute maximum λmax of individual target
compounds. All NMR spectra were recorded on a Bruker AM-series, Bruker BioSpin Corp.,
Germany. The working frequency is given for each compound. Chemical shifts are reported
in ppm (δ) against the internal standard, Si(CH3)4. Easily exchangeable signals were omitted
when diffuse. Signals are designated as follows: s, singlet; d, doublet; dd, doublet of doublets; t,
triplet; m, multiplet; bs, broad singlet.
Compounds 1a, 1b, and 3a were synthesized according to the literature [17].
Preparation of nanosized palladium particles on the copper (5% Pd/Cu). A solution of
methanol (1050 mL) and aqueous ammonia (25 wt. %, 370 mL) was stirred for 15 min, fol-
lowed by the addition of tetraethyl orthosilicate (75.09 mL). The reaction mixture was vigor-
ously stirred for 2 h at room temperature to produce the colloidal silica suspension. The
resultant colloidal silica suspension was centrifuged. Deionized water (100 mL) was added to
the colloidal silica and the mixture was placed in an ultrasound bath and sonicated for 20 min.
Then, an aqueous saturated solution containing 1.72g PdCl2 was added dropwise into the sus-
pension of colloidal silica and mixed in an ultrasound bath for 20 min. The mixture was dried
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Fig 1. Structures of the quinoline derivatives.
doi:10.1371/journal.pone.0131210.g001
at 60–90°C for 12 h, ground and sieved. Finally, the reduction was conducted in an oven under
hydrogen at 500°C for 1 h to produce nanosized metallic particles that were dispersed on the
SiO2 intermediate carrier. Nanosized palladium particles were dispersed on the SiO2 (0.55 g)
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
and Cu (0.5 g) was added to the deionized water (100 mL) and stirred for 10 min. Then, a
freshly prepared sodium hydroxide solution (80 mL 40% w/w) was added and the suspension
was stirred for 1 h. Next, the suspension was cooled to room temperature and allowed to form
sediment. After decantation, the sediment was washed to neutral pH with deionized water. The
resulting preparation of the catalyst was dried to a constant mass at room temperature [18].
General method synthesis of substrates 2b and 2c In a tightly sealed tube (septa system),
aryl halides (5.5 mmol) and 5% nanocatalyst Pd/Cu, PPh3 (17 mg) were suspended in dry
triethylamine (10 mL). The mixture was placed in an ultrasound bath and sonicated for 5 min.
Then, the acetylene compound (5.6 mmol) was added and the mixture was stirred for 3 h. The
mixture was cooled to room temperature and the catalyst was centrifuged, filtered and washed
with ethyl acetate (3 x 10 mL). The filtrate was washed three times with deionized water (3 x 15
mL) and then dried over magnesium sulfate, filtered and concentrated under reduced pressure
to give the product.
Synthesis of 4-[(trimethylsilyl)ethynyl)benzaldehyde (2b): The product was obtained in a
quantitative yield, melting point 62–63°C. 1H NMR (400 MHz, DMSO-d6) δ: 10.02 (s, 1H),
7.90 (d, J = 8.55 Hz, 2H), 7.67 (d, J = 8.10 Hz, 2H), 0.26 (s, 9H). 13C NMR (101 MHz, DMSO-
d6) δ: 192.34, 136.20, 132.74, 130.04, 127.83, 104.57, 99.21, 0.18.
Synthesis of 4-(phenylethynyl)benzaldehyde(2c): The product was obtained in a quantitative
yield, melting point 95–98°C. 1H NMR (400 MHz, CDCl3): δ 10.40 (s, 1H), 7.89 (d, J = 8.2 Hz,
2H), 7.70 (d, J = 8.10 Hz, 2H), 7.58 (m, 2H), 7.40 (m, 3H). 13C NMR (101 MHz, CDCl3) δ
191.41, 135.44, 132.12, 131.81, 129.59, 128.98, 128.49, 122.52, 93.47, 88.53.
1. Compound 3b. 0.4 mL quinaldine (3 mmol) was dissolved in 10 mL of acetic anhydride
with 3 mmol of aldehyde 2b. The resulting mixture was stirred under argon at 130°C for 20 h.
After evaporation to dryness, the product was purified by a short SiO2 column (eluent–ethyl
acetate/hexane). The solution was then cooled and concentrated under reduced pressure. Next,
the mixture was washed with diethyl ether. The product was obtained as a yellow solid with
yields of 55%, melting point 191°C. 1H NMR (400 MHz, DMSO-d6) δ: 8.15 (d, J = 8.6 Hz, 1H),
8.12 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.77–7.71 (m, 1H), 7.67 (d, J = 8.7 Hz, 2H),
7.59 (d, J = 8.3 Hz, 2H), 7.55–7.50 (m, 3H), 7.43 (d, J = 16.3 Hz, 1H), 0.30 (s, 9H). 13C NMR
(101 MHz, DMSO-d6) δ: 155.80, 148.17, 137.34, 137.00, 33.53, 132.64, 130.56, 130.32, 129.22,
128.26, 128.06, 127.89, 127.60, 126.78, 122.59, 120.60, 105.69, 96.08, 0.28, MS (EI) m/z:
[M+H]+ Calcd for C22H21NSi 328.16; Found 328.35.
2. Compound 3c. K2CO3 was added to 3b dissolved in MeOH and the resulting mixture
was stirred for 2 h at 30°C. The mixture was concentrated and added to water/diethyl ether and
further extracted with diethyl ether. The organic layers were washed with water and brine and
dried over MgSO4. After the evaporation of the solvent, a yellow solid was obtained with a
yield of 90%, melting point 154°C. 1H NMR (400 MHz, DMSO-d6) δ: 8,16 (d, J = 8.6 Hz, 1H),
8.11 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.76–7.71 (m, 1H), 7.70–7.65 (m, 2H), 7.62 (d,
J = 8.3 Hz, 2H), 7.56–7.51 (m, 3H), 7.44 (d, J = 16.3 Hz, 1H), 3.19 (s, 1H), 13C NMR (101 MHz,
DMSO-d6) δ: 155.58, 148.26, 137.00, 136.48, 133.47, 132.56, 130.04, 129.86, 129.25, 127.49,
127.44, 127.11, 126.36, 122.13, 119.43, 83.60, 78.39, MS (EI) m/z: [M+H]+ Calcd for C19H13N
256.12; Found 256.28. Anal. Calcd for C19H13N: C, 89.38; H, 5.13; N, 5.49. Found: C, 89.64; H,
5.47; N, 5.32.
3. Compound 3d. 0.4 mL quinaldine (3 mmol) was dissolved in 10 mL of acetic anhydride
with 3 mmol of aldehyde 2c. The resulting mixture was stirred under argon at 130°C for 20 h.
After evaporation to dryness, the product was purified by a short SiO2 column (eluent–dichlor-
omethane). The solution was concentrated under reduced pressure. Next the mixture was
washed with diethyl ether. The product was obtained as a pale yellow solid with yields 50%,
melting point 201–203°C. 1H NMR (400 MHz, CDCl3) δ: 8.15 (d, J = 8.6 Hz, 1H), 8.12 (d,
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
J = 8.5 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.77–7.71 (m, 1H), 7.67 (dd, J = 13.7, 8.4 Hz, 4H),
7.61–7.56 (m, 4H), 7.53 (t, J = 7.5 Hz, 1H), 7.45 (d, J = 16.3 Hz, 1H), 7.41–7.35 (m, 3H). 13
NMR (101 MHz, CDCl3) δ: 155.70, 148.29, 136.44. 133.65, 132.03, 131.65, 129.83, 129.74,
C
129.26, 128.39, 127.53, 127.43, 127.20, 126.31, 123.39, 123.23, 119.42, 90.84, 89.46, MS (EI) m/
z: [M+H]+ Calcd for C25H17N 332.14; Found 332.33. Anal. Calcd for C25H17N: C, 90.60; H,
5.17; N, 4.23. Found: C, 90.35; H, 5.11; N, 4.40.
B. Cell culture
Human colon adenocarcinoma cells (HCT116) and normal human fibroblast (GM 07492) cells
were obtained from the American Type Culture Collection. Cells were grown as monolayer
cultures in 75 cm3 flasks (Nunc) in Dulbecco's Modified Eagle Medium (DMEM). The medium
was supplemented with 12% heat inactivated fetal bovine serum (PAA) and for GM 07492
with 15% fetal bovine serum (Gibco) and 100 μg/mL of gentamycin (Gibco). The cell lines
were maintained at 37°C in a 5% CO2 incubator and passaged every 3–4 days as required.
C. Cytotoxicity assay
Exponentially growing cells were harvested through the trypsinization of sub-confluent cul-
tures. Cells were seeded into 96-well cell culture microtiter plates (Nunc) at concentrations of
5.0 x 103 cells per well and cultured for 24 h. After this time, the growth medium was
exchanged for a medium containing the compounds in concentrations ranging from 0.5 μM-
25 μM. Stock solutions of the compounds being investigated were prepared in sterile DMSO.
The final concentration of DMSO in the medium did not exceed 0.2%. After a 72 h incubation
with the compounds being investigated under standard cell culture conditions, the medium
was replaced with 100 μL of DMEM without phenol red. The metabolic activity of viable cells
was determined by adding 20 μL of CellTiter 961AQueousOne Solutions–MTS (Promega) to
each well followed by a 1 h incubation. The MTS assay is a colorimetric method for determin-
ing the number of viable cells. A standard solution containing 100 μL of DMEM without phe-
nol red and 20 μL of MTS solution was used to determine”blank” absorbance. The absorbance
was measured at 490 nm using a SynergyTM4 microplate reader (BioTek). The inhibitory con-
centration (IC50) was defined as the compound concentration that was necessary to reduce the
proliferation of cells to 50% of the untreated control cells and expressed as means standard
deviation (SD) in GraphPad Prism 5 software. Each individual compound was tested in tripli-
cate in a single experiment with each experiment being repeated 3–5 times.
D. The cellular staining
To visualize the accumulation of the compounds within the cells, 10×103 HCT116 cells in a
300 μL growth medium were plated into an 8-well LabTek chambered cover glass (Nunc) and
incubated under standard conditions at 37°C in a humidified atmosphere at 5% CO2 for 24h.
After this, the cells were treated with the compounds being tested (3a, 3c and 3d) at a concen-
tration of 25 μM and incubated for a further 2h. After incubation the cells were washed three
times with PBS and then 300 μL of PBS was added. Observation of the cells was carried out
using an inverted fluorescence microscope (IX81, Olympus) immediately after staining.
E. Spectroscopic studies
The absorption and fluorescence spectra were measured at room temperature in a 10 mm
quartz cell with a U-2900 spectrophotometer (Hitachi) and an F-7000 spectrofluorimeter
(Hitachi), respectively. The styrylquinoline stock solutions (10mM) that had been prepared in
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
DMSO were further diluted with appropriate spectroscopic grade solvents to working concen-
trations starting from0.1mM (1% DMSO).The fluorescence quantum yields were measured
using the comparative method with anthracene in cyclohexane as a reference (ϕref = 0.34) [19].
We prepared standard and test solutions of decreasing concentrations in order to provide
absorbance in the range of 0.1 to 0.02 at the excitation wavelength. The fluorescence spectra of
all of the solutions were measured at room temperature in a 10 mm cell using an F-7000
spectrofluorimeter (Hitachi). The fluorescence quantum yields of the dyes that were tested
were calculated as follows: ꢀdye ¼ ꢀref ꢀ ðadye=aref Þ ꢀ ðZ2dye=Zr2ef Þ, where subscripts dye and ref indi-
cate test and standard samples, respectively, ϕ is the fluorescence quantum yield, α is the gradi-
ent obtained from the plot integrated fluorescence intensity versus absorbance and η is the
refractive index of solvents that were applied.
F. Calculations
DFT and TD-DFT calculations for styrylquinoline derivatives were performed using the
Gaussian 09 software package [20] with the B3LYP exchange-correlation functional [21] and
6–31+G(d,p) basis set. The effects of the solvent were evaluated using the PCM model[22–24]
in which a cavity is created via a series of overlapping spheres [25] with standard dielectric con-
stants (ε) of 46.826 for DMSO. Electronic absorption transition energies for 32 spin-allowed
singlet-singlet (S0!Sn) and fluorescence emission spectra made of the 8 spin-allowed Sn!S0
transitions were considered using the external iteration (EI) approach [26–29]. The molecular
electron densities for each derivative were determined from the wave functions using the
CUBE option implemented in Gaussian 09 and visualized using GaussView 5.0. The molecular
energy levels were analyzed using Chemissian software.
Results and Discussion
A. Synthesis and characterization
Our synthetic approach is depicted in Fig 2. The compounds that were investigated were pre-
pared using the Sonogashira reaction in homogeneous or heterogeneous conditions.
Compounds 3a, 3b, 3d were obtained according to the standard method [9, 10, 30] from
quinaldine and aldehydes 2a-c in acetic anhydride, while aldehydes 2b-c were obtained in the
Sonogashira coupling in heterogeneous conditions on a nano-Pd catalyst. For the preparation
of the bimetallic catalyst 5% Pd/Cu, we modified a previously described method [18].The over-
all activity of the nano-particle catalysts depends mainly on the effectiveness of the distribution
of the metallic grains in the matrix [31]. The bimetallic nano-Pd/Cu was obtained by digesting
the carrier in nano-Pd/silica and transferring the nanoparticles on to the electrolytic copper.
We found that digestion of the material dispersed in an excess of the NaOH resulted in a short-
ened time of the procedure and reduced the temperature. Such prepared nanoparticles of the
catalyst retain their high activity[18]. This catalytic system provided us with quantitative con-
versions. Compound 3c was obtained through the hydrolysis of 3b immediately after its syn-
thesis as is shown in Fig 3. All of the compounds that were obtained were characterized using
NMR and mass spectroscopy.
B. Biological activity
The antiproliferative activity of the compounds that were synthesized was assessed using the
MTS assay. The results from the cytotoxicity assay are shown in Table 1. In general, the com-
pounds that were tested appeared to be inactive against the HCT116 cell lines.
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Fig 2. Sonogashira cross-coupling reaction and synthesis of SQLs. The direct alkylation of the 2-bromoquinoline produced analogs of
styrylquinolines1a-b [17].
doi:10.1371/journal.pone.0131210.g002
Fig 3. Hydrolysis of compound 3b.
doi:10.1371/journal.pone.0131210.g003
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Table 1. Antiproliferative activity.
com.
IC50 [μM]
HCT116
GM 07492
1a
1b
3a
3c
3d
>25a
>25
>25
>25
>25
>25
>25
21.18 3.36
>25
>25
aResults are expressed as mean standard deviation from 3–5 experiments.
doi:10.1371/journal.pone.0131210.t001
All of the tested compounds were also examined for their cytotoxic effects against the nor-
mal human fibroblasts. All of the compounds that were tested proved to be inactive. In the con-
text of the potential use of these compounds as fluorescent dyes, the lack of cytotoxicity is a
significant advantage.
C. Cellular imaging
The excitation waves for compounds that were tested are relatively short, which can cause
problems in biological applications. The sample autofluorescence and poor background sup-
pression are the most important here. Nevertheless, we decided to perform the appropriate
experiments and got some interesting results. For the staining experiments, we selected the
most effective compounds 3a, 3c and 3d. The cellular staining of selected compounds in the
human colon carcinoma cells (HCT116) is presented in Fig 4. After a 2h incubation, all of the
compounds efficiently penetrated the cellular membranes and a strong blue signal was
Fig 4. Live cell imaging of the HCT116 cells following treatment with 3a, 3c and 3d using bright field optical microscopy (BF) and fluorescence
microscopy (DAPI filter).
doi:10.1371/journal.pone.0131210.g004
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
observed. Interestingly, 3a (t-butyl derivative) seems to locate primarily in plasma membrane
rather than in the internal organelles. On the other hand, compounds 3c (R = H) and 3d
(R = Ph) accumulated intracellularly. The micrographs of these two compounds are compara-
ble to lysosome staining. The localization of the 3c and 3d may be partially explained based on
the physicochemical properties of the tested compounds. Styrylquinolines are weak bases and
their lipophilicity is relatively high (AlogP: 3a: 6.31, 3c: 4.73, 3d: 6.51). Such compounds gener-
ally exhibit lysosomotropism. The predominant localization of the 3a in plasma membrane
remains unclear.
D. Fluorescent properties
The absorption and emission properties of the compounds that were tested were measured in
several solvents that had decreasing polarity (DMSO > ethanol > chloroform). The absorption
spectra that were registered did not show any evident solvent-dependent spectral shifts (see
Table 2). In general, the spectrum of 1b was the one that shifted most to shorter wavelengths.
All of the compounds have relatively high values of molar absorption coefficients (ε) (see
Table 2). Interestingly, we observed that the ε values were significantly higher in DMSO than
in other solvents. The highest ε value was observed for 3d in DMSO (ε = 44 000 M-1cm-1 at
360 nm).
The emission spectra of the compounds that were synthesized covered the colors from violet
to blue. For 1a in the DMSO solution, the maximum fluorescence emission was observed in
the ultraviolet region (λem = 367 nm) while for the others the maximum was blue-shifted and
was observed in the visible region of the electromagnetic spectrum (λem = 407–411 nm, see Fig
5-B). For solvents that had a lower polarity, the emission maxima were slightly shifted towards
shorter wavelengths. In the case of 1b the weak fluorescence emission was observed only for
solutions prepared in ethanol.
Table 2. Fluorescent and absorption properties of quinolone dye solutions in various solvents.
λmax [nm](εꢀ103 [M-1 cm-1])
λem [nm]
Stokes shift [nm]
ϕdye
DMSO
1a (wk39)
1b (wk40)
3a (wk44)
3c (mk7)
3d (mk9)
Ethanol
278 (17.9)
288 (0.51)
260 (37.0)
342 (33.8)
360 (44.0)
367
-
23
-
0.01
-
407
408
411
47
52
51
0.06
0.01
0.32
1a (wk39)
1b (wk40)
3a (wk44)
3c (mk7)
3d (mk9)
Chloroform
1a (wk39)
1b (wk40)
3a (wk44)
3c (mk7)
3d (mk9)
279 (14.8)
333(5.91)
347 (18.4)
344 (18.7)
356 (24.5)
369
350
399
390
412
23
17
40
32
56
0.19
0.01
0.06
0.04
0.24
280 (14.7)
332 (6.58)
347 (18.4)
344 (18.5)
357 (24.1)
361
-
15
-
0.11
-
400
389
410
38
30
50
0.06
0.03
0.38
doi:10.1371/journal.pone.0131210.t002
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Fig 5. UV-VIS absorption (A) and normalized fluorescence (B) spectra of styrylquinolines solutions in DMSO.
doi:10.1371/journal.pone.0131210.g005
The Stokes shift reflects a loss of energy between the excitation and emission that was
observed in the solution. As a result, the emission spectrum is naturally shifted toward longer
wavelengths. A small value of the Stokes shift can negatively affect the sensitivity of fluores-
cence detection. The differences that were observed between excitation and emitted light for
the dyes described in this report are applicable (15–56 nm, see Table 2).
The fluorescence quantum yield is one of the key parameters of fluorophores and permits a
quantitative description of the fluorescence phenomenon. It is defined as the number of pho-
tons being emitted relative to the number of photons being absorbed. The quantum yields that
were calculated are presented in Table 2. The strong influence of solvent on fluorescence emis-
sion was observed for 1a (Fig 6). Changing the solvent from DMSO to ethanol and chloroform
led to a noticeable growth of fluorescence efficiency from 0.01 to 0.19 and 0.11, respectively.
Among the compounds that were investigated, 3d had a significantly higher quantum
Fig 6. The comparison of emission spectra of 1a in solvents of various polarity
doi:10.1371/journal.pone.0131210.g006
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
efficiency than the others (e.g., 0.38 in chloroform), which means that incorporating an addi-
tional aromatic unit positively affects the fluorescent properties of a fluorophore. The same
seems to be true for compounds 1a and 1b for which only a phenyl substituted structure
revealed some fluorescent potency. This is in agreement with the general opinion about the
nature of the fluorophoric properties of the aromatic core. Polycyclic systems and scaffolds
packed with π conjugated bonds are the base of the most effective fluorophores. Recently,
Yamaguchi and coworkers presented an apparent correlation between the structure of π conju-
gated hydrocarbons and their quantum yield of fluorescence [32]. According to that report,
both the fluorescence emission maximum and quantum yield are higher in longer π conjuga-
tion in an S1 state. Namely, the longer the linear donor/acceptor conjugated dipole the better
its fluorescent properties. The compounds that were tested, although unsubstituted on account
of their biological indifference, should follow this rule to some extent. To evaluate this, we per-
formed some additional calculations as follows.
E. Theoretical calculations
To better understand and characterize the absorption spectra of the compounds that were
tested, TD-DFT calculations were performed with B3LYP exchange-correlation functional and
6–31+G(d,p) basis set.
The most important parameters such as transition energies, oscillator strengths (f) and the
main configurations are listed in Table 3along with experimental absorption data. The rest of
the electronic transitions were omitted due to the very low intensity of parameter f and were
not taken into account during further data analysis.
The applied theoretical approach correctly predicted the experimental maximum absorp-
tion data for 1a, 3a, 3c and 3d. On the other hand, the experimental bands for 1b were not
fully reflected by theory. This could be due to the geometry of the molecule. In general, these
calculations confirmed that the number of double and triple bonds shifts the absorption maxi-
mum to longer wavelengths (i.e. bathochromic shift). This observation was further confirmed
with the HOMO/LUMO orbitals and energy gap (ΔEH-L) analysis according to the scheme pre-
sented in the literature [33, 34]. The theoretical and experimental data are summarized in
Table 4. It has been found that due to the growing energy gap (ΔEH-L), the level of conjugation
decreases in the following order 3d!3a!3c!1a!1b. In conjugated molecules, the energy
gap between HOMO and LUMO orbitals is lower, which increases the possibilities of electron
promotion at longer waves. This is reflected in our results as the experimental absorption wave-
lengths that were observed for 3d and 3a are more red-shifted with respect to the least-coupled
system – 1b.
According to the theoretical data, the experimental absorption maxima for 3c and 3d are
characterized by S0!S1 (assigned to π!πꢁ) transitions that are composed of HOMO!LUMO
configurations (see Table 3). The presence of a tert-butyl group in molecule 3a prevents π-con-
jugation, and consequently, the S0!S1 electronic transition is a mixture of HOMO!LUMO
(69%) and HOMO-1!LUMO (13%) configurations.
In the case of 1b the low intensity experimental bands corresponded with the theoretical
bands due to the S0!S3 and S0!S4 electronic transitions, while the most intensive experimen-
tal bands (not reflected by theory) would be associated with S0!S1 and S0!S2. It is noteworthy
that the isosurfaces of the HOMO and LUMO orbitals are localized through the central double
bond and the double bonds of the rings in all of the molecules (Fig 7), while LUMO distribu-
tion is also localized on the nitrogen.
The higher electronic S0!Sn (n = 2, 3, 4) (π!πꢁ) transitions for almost all of the systems
generally consisted of mixed HOMO-1!LUMO and HOMO!LUMO+1. Another character is
PLOS ONE | DOI:10.1371/journal.pone.0131210 June 26, 2015
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Table 3. Electronic transition data obtained by EI TD-DFT/B3LYP/6-31+G(d,p) using a PCM model (solvent–DMSO) for quinoline dyes at the DFT
optimized geometry.
com.
1a
Electronic transitions
Theoretical λmax[nm]
f
molecular orbital(MO)
% coefficient
experimental λmax[nm]
S0!S1
S0!S2
335.9
318.3
0.997
0.012
HOMO!LUMO
HOMO-1!LUMO
HOMO!LUMO+1
HOMO-1!LUMO
HOMO!LUMO+1
-
68
62
27
28
60
-
344
330
S0!S4
281.2
0.585
278
1b
-
-
-
330
318
304
-
-
-
-
-
S0!S3
310.6
0.193
HOMO!LUMO
HOMO!LUMO+1
HOMO-1!LUMO
HOMO!LUMO
HOMO!LUMO+1
HOMO!LUMO
HOMO-1!LUMO
HOMO-1!LUMO
HOMO!LUMO
HOMO-3!LUMO
HOMO!LUMO+1
HOMO!LUMO+2
HOMO!LUMO
HOMO-1!LUMO
HOMO!LUMO+1
HOMO-1!LUMO
HOMO!LUMO
HOMO-1!LUMO
62
20
57
27
28
69
13
67
11
41
25
39
70
55
39
41
70
28
S0!S4
293.3
0.021
288
3a
S0!S1
S0!S2
S0!S6
381.4
357.1
292.4
1.041
0.364
0.253
360
346
292
3c
3d
S0!S1
S0!S2
377.8
329.6
0.949
0.063
356
342
S0!S4
S0!S1
S0!S3
295.9
401.9
309.5
0.504
1.632
0.298
290
360
305
doi:10.1371/journal.pone.0131210.t003
observed for 3a where the second-lying S0!S2 transition is due to two molecular orbital contri-
butions (HOMO-1!LUMO + HOMO!LUMO) and the lowest-lying S0!S6 transition corre-
sponded to the HOMO-3!LUMO + HOMO!LUMO+1 + HOMO!LUMO+2 excitations.
Moreover, the atomic orbitals of the carbon of the double bond provide a significant contribu-
tion to HOMO but no contribution to HOMO-1. In the case of LUMO+1, the electron distribu-
tion was concentrated on the atoms of the pyridine and phenyl ring. Noticeably, electronic
S0!Sn (n = 3, 4) (π!πꢁ) transitions for 1b consisted of mixed HOMO-1!LUMO and
HOMO!LUMO+1 as well as HOMO–LUMO molecular orbitals.
Table 4. Negative orbital (au) of the HOMO (-EHOMO) and LUMO energies (-ELUMO), HOMO–LUMO band gap energies that were calculated by PCM//
DFT-DFT/B3LYP/6-31+G(d,p) using the PCM model (solvent–DMSO) and the theoretical excited energies (Eg) for dyes.
com.
-EHOMO1
-EHOMO
-ELUMO
-ELUMO+1
ΔEH-L[eV] (DFT)
Eg[eV] (TD-DFT)
1a
1b
3a
3c
3d
6.77
6.86
6.65
6.62
6.59
6.52
6.55
5.88
6.49
5.82
2.16
2.04
2.38
2.29
2.48
1.39
1.15
1.40
1.44
1.62
4.36
4.51
3.50
4.19
3.33
3.39
3.96
3.25
3.28
3.08
doi:10.1371/journal.pone.0131210.t004
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Fig 7. Molecular orbitals of the five quinoline derivatives obtained at the TD-B3LYP/6-31+G(d,p) theory level.
doi:10.1371/journal.pone.0131210.g007
The molecular orbital analysis confirmed that the difference between styrylquinoline prop-
erties depends on the conjugation. The orbital energy levels increase for HOMO
(1b!1a!3c!3a!3d) and LUMO (3d!3a!3c!1a!1b) with respect to the number of
conjugated groups. This relationship is clear from the point of view of the electronic properties
of the molecular fragments with alkyne group (CꢂC–H; 3c), which are the most electron-with-
drawing. On the other hand, the strong electron-donating and activating phenyl group that
was attached to the CꢂC fragment of 3d molecule makes this compound the most nucleophilic
of all those studied.
The fluorescence emission energies for the spin-allowed singlet-singlet (Sn!S0) transitions
were calculated for the dye solutions in DMSO using the external iteration (EI) TD-DFT//
B3LYP/6-31+G(d,p) approach. The transition energies, oscillator strengths and main configu-
rations that were computed for the most relevant excited states of each molecule were com-
pared with the experimental values and are summarized in Table 5.
The wavelengths of the maximum emission that were calculated are in good agreement with
the experimental values for all of the compounds. The theoretical emission value for 1b was
found to be very low despite the lack of the luminescence effect that was observed in the experi-
ment. This may be the effect of the quenching process as the change of the solvent revealed
some fluorescence (see Table 2). The theoretical data for 3a explained the experimental emis-
sion peak as a mix of S2!S0 + S3!S0 electronic transitions within HOMO!LUMO and
HOMO-2!LUMO (see Table 5).
The extinction coefficients that were obtained from the theoretical outcomes correspond
with the experimental quantum yields (Fig 8). This correlation indicated that such a theoretical
approach may be a simple and convenient tool for predicting the emission features of newly
synthesized systems. As was expected, the more conjugated system had a higher emission
intensity and quantum yield. Furthermore, the introduction of a strongly electron-donating
PLOS ONE | DOI:10.1371/journal.pone.0131210 June 26, 2015
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Table 5. Emission data obtained by EI TD-DFT/B3LYP/6-31+G(d,p) using the PCM model (solvent–DMSO) for SQLs at the DFT optimized geometry.
com.
1a
Electronic transitions
theoreticalλem[nm]
f
Molecular orbital (MO)
% coefficient
εemꢀ103
2
experimental λem[nm]
S1!S0
401.7
0.017
HOMO!LUMO
HOMO-1!LUMO
HOMO!LUMO
HOMO-1!LUMO
HOMO!LUMO+1
HOMO!LUMO
HOMO-2!LUMO
HOMO!LUMO
HOMO-2!LUMO
HOMO!LUMO
HOMO!LUMO
50
48
70
66
22
17
69
17
66
70
71
-
1b
3a
S1!S0
S2!S0
362.7
349.5
0.835
0.138
37
6
367
407
S2!S0
S3!S0
419.9
385.9
1.039
0.485
52
24
3c
3d
S1!S0
S1!S0
421.1
434.9
1.487
1.919
48
75
402
411
doi:10.1371/journal.pone.0131210.t005
group (e.g., benzene) in the alkyne linker–CꢂC–significantly enhanced both of the features
mentioned above.
Fig 8. Theoretical extinction coefficient compared with the experimental quantum yield. Green arrows: a system with poor conjugation (1a, 1b), yellow
arrows: strongly conjugated molecules (3a, 3c, 3d).
doi:10.1371/journal.pone.0131210.g008
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Quinoline Dyes from Nanocatalyzed Sonogashira Coupling
Conclusions
A series of five quinoline derivatives were designed based on the styrylquinoline system. These
compounds were obtained according to the novel method of Sonogashira coupling on a
bimetal nanocatalyst with a satisfactory to good yield. The preparation of the nanocatalyst was
also improved according to known methods, which substantially improve the availability of
this system. The compounds that were obtained were tested for their biological activity against
human cancer cells and normal fibroblasts. Their absorption and fluorescent spectra were col-
lected with quantum yields. DFT and TD-DFT calculations were carried out to confirm the
experimental observations. The compounds that are presented have a low toxicity and are tol-
erable in biological systems in useful concentrations for a long time (72 h vs 2 h observation).
Their fluorescent properties make them potentially interesting leading structures for further
development. Moreover, the theoretical calculation that is presented allows the direction of the
modification of basic compounds to intensify its luminescence parameters to be proposed.
Acknowledgments
The financial support of the National Center for Science NCN grants 2014/13/D/NZ7/00322
(A.M.W.), 2012/07/N/NZ7/02110 (W.C.), 2013/09/B/NZ700423 (R.M.) and DEC-2012/07/N/
ST5/02221 (M.D.) is greatly appreciated. E.S. and M.K. appreciate the DoktoRIS studentship
and W.C. and M.D. appreciate the CITTRFUŚ fellowship. Nano-Pd/Cu was prepared under
National Research and Development Center (NCBiR) Grant ORGANOMET no: PBS2/A5/40/
2014. This research was supported in part by PL-Grid Infrastructure.
Author Contributions
Conceived and designed the experiments: MRB AMW RM. Performed the experiments: MRB
MD WC AMW MK ES. Analyzed the data: AMW MRB RM. Contributed reagents/materials/
analysis tools: AR PB. Wrote the paper: MRB RM ES JP.
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