Azaindole-BODIPYs: Synthesis, fluorescent recognition of hydrogen sulfate anion and biological evaluation

Article abstract of DOI:10.1016/j.saa.2019.01.047

The synthesized and sensing capability of two novel azaindole substituted mono and distyryl BODIPY dyes against bisulfate anion were reported. Structural characterizations of the targeted compounds were conducted by using matrix-assisted laser desorption/

Full text of DOI:10.1016/j.saa.2019.01.047

Accepted Manuscript
Azaindole-BODIPYs: Synthesis, fluorescent recognition of
hydrogen sulfate anion and biological evaluation
Gürkan Keşan, Burcu Topaloğlu, Emrah Özcan, Hasan Hüseyin
Kazan, Esra Tanrıverdi Eçik, Elif Şenkuytu, Ibrahim F. Sengul,
Hakan Kandemir, Bünyemin Çoşut
PII:
S1386-1425(19)30056-3
https://doi.org/10.1016/j.saa.2019.01.047
SAA 16733
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 17 July 2018
Revised date:
Accepted
date:
7 December 2018
1
5 January 2019
Please cite this article as: Gürkan Keşan, Burcu Topaloğlu, Emrah Özcan, Hasan Hüseyin
Kazan, Esra Tanrıverdi Eçik, Elif Şenkuytu, Ibrahim F. Sengul, Hakan Kandemir,
Bünyemin Çoşut , Azaindole-BODIPYs: Synthesis, fluorescent recognition of hydrogen
sulfate anion and biological evaluation. Saa (2019), https://doi.org/10.1016/
j.saa.2019.01.047
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ACCEPTED MANUSCRIPT
Azaindole-BODIPYs: Synthesis, Fluorescent Recognition of
Hydrogen Sulfate Anion and Biological Evaluation
a
a
a
b
Gürkan Keşan , Burcu Topaloğlu , Emrah Özcan , Hasan Hüseyin Kazan , Esra Tanrıverdi
a
a
a
c
a*
Eçik , Elif Şenkuytu , Ibrahim F. Sengul , Hakan Kandemir , Bünyemin Çoşut
Contribution from:
a
Department of Chemistry, Faculty of Science, Gebze Technical University, Gebze, Kocaeli,
Turkey
b
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey
cDepartment of Chemistry, Faculty of Art and Science, Namık Kemal University, Tekirdag,
Turkey
*Author for correspondence:
Dr. Bünyemin Çoşut, Department of Chemistry, Gebze Technical University, P.O.Box: 141,
Gebze 41400, Kocaeli, Turkey
Tel:
00 90 262 6053015
00 90 262 6053105
bc@gtu.edu.tr
Fax:
e-mail:

ACCEPTED MANUSCRIPT
Abstract
The synthesized and sensing capability of two novel azaindole substituted mono and distyryl
BODIPY dyes against bisulfate anion were reported. Structural characterizations of the
targeted compounds were conducted by using matrix-assisted laser desorption/ionization
1
13
time-of-flight mass spectrometry, H and C NMR spectroscopies. Photophysical properties
of the azaindole substituted BODIPY compounds were investigated employing absorption and
fluorescence spectroscopies in acetonitrile solution. It was found that the final compounds 3
and 4 exhibited exclusively selective and sensitive turn-off sensor behavior on HSO -
anion.
4
Additionally, the stoichiometry ratio of the targeted compounds to bisulfate anion was
measured 0.5 by Job’s method. Also, density function theory was performed to the optical
response of the sensor for targeted compounds. Furthermore, the cytotoxicity of Azaindole-
BODIPYs were examined against living human leukemia K562 cell lines.
Keywords: Borondipyrromethenes, Azaindole, hydrogen sulfate, Anionsensor, Fluorescence,
Chemosensor, K562 cell lines, DFT.
2

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1
. Introduction
The construction of cation and anion fluorescent chemosensors has a growing interest over the
past years since they play a critical role in chemistry fields related with organic,
supramolecular and biological process [1-5]. The extensive researches on metal and pH
sensors which have been previously investigated showed that they are important in both
biological and environmental processes [6,7]. Particularly, among the variety of anion
sensors, the detection of bisulfate (HSO -
) anions are extremely important. Firstly, they are
4
found in many agricultural fertilizers, industrial raw materials, nuclear fuel waste and toxic
effect in pollutant [8]. Secondly, in the point of their biological applications, HSO -
dissociate at high pH to generate toxic sulphate (SO -2
4
anions
4
) ions. These toxic ions cause irritation
of the skin, eyes and respiratory system [9,10]. Despite this importance, only a few examples
have been reported in the literature for detection of HSO -
4
anions [11-15].
The design of fluorescent sensors can be arranged by covalently integrated photoactive
fragment(s) with a receptor subunit(s). In this sense, the units of the fluorescent sensors play a
critical role for detection of anions. On the one part, as a photoactive fragment in fluorescent
sensors, Boron dipyrromethane (BODIPY) has been attractive targets within highly
fluorescent dyes over the last few decades [16]. They frequently display an excellent
photophysical properties such as high fluorescence quantum yield, large extinction
coefficient, high chemical, thermal and photophysical stability [17-20]. Thus, they have a
wide range of applications include fluorescent biolabelling reagents, light harvesting
materials, optical chemosensor and photodynamic therapy reagents [21-25]. And therefore,
structures containing BODIPY scaffolds present fluorescent sensor for both anions and
cations [26,27]. On the other part, as a receptor subunit, the azaindole nuclei which have
nitrogen containing aromatic heterocyclic compounds can be used. They have a great
attention because of their valuable physicochemical and pharmacological properties with
3

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potential application in medicinal chemistry [28-31]. Thus, the combinations of BODIPY and
azaindole nuclei can be useful for many applications in chemistry.
Covalently combined BODIPY core and azaindole moieties were reported by
Mahapatra et al. [32]. In their study, an azaindole motif placed at the meso (8) position of
1
BODIPY core was investigated by H NMR, steady-state and emission spectroscopies,
suggesting a dual channel detection of fluoride (absorption) and AcO- anions (emission)
among various anions. In the present work, we are interested in the combination of BODIPY
and azaindole motifs which is at only 3-position (mono) and, both 3- and 5- positions (distryl)
of BODIPY (Scheme 1, compound 3 and 4, respectively) core to investigate their
photophysical and anion selectivity properties. It is shown here that the difference in
azaindole position with respect to BODIPY core affects the π-electron distributions. However,
newly developed compounds enable to detect HSO -
anion. Also, we report here in vitro
4
cytotoxicity effects against Human leukemia K562 cell line for titled compounds.
Scheme 1 Chemical structure and synthetic pathway of Azaindole-BODIPYs (3 and 4).
4

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2
2
. Experimental
.1. Materials
The deuterated solvent (CDCl ) for NMR spectroscopy, silica gel, dichloromethane
3
and metal chlorides were provided from Merck. Following chemicals were obtained from
Sigma Aldrich; 2,4-dimethylpyrrole, benzaldehyde, trifluoroacetic acid, p-Chloranil,
triethylamine, Boron trifluoride diethyl etherate, glacial acetic acid, piperidine, hydrogen
peroxide, N,N-dimethylformamide, benzene and 1,8,9-Anthracenetriol for the MALDI matrix
was obtained from Fluka. All other chemicals used for the synthesis were reagent grade unless
−
−
2−, HPO
2−, HSO −
, and CH
COO-).
otherwise specified like sodium anions (I , F , SO
4
4
4
3
2
.2. Equipment
Electronic absorption spectra were recorded with a Shimadzu 2101 UV
spectrophotometer in the UV-visible region. Fluorescence excitation and emission spectra
were recorded on a Varian Eclipse spectrofluorometer using 1 cm pathlength cuvettes at room
temperature. The reversibility measurements of the probes were conducted with UV
spectrophotometer and Varian Eclipse spectrofluorometer by using 0.1M H
2
2
O . The
fluorescence lifetimes were obtained using Horiba- Jobin-Yvon-SPEX Fluorolog 3-2iHR
instrument with Fluoro Hub-B Single Photon Counting Controller at an excitation wavelength
of 470 nm. Signal acquisition was performed using a TCSPC module. Mass spectra were
acquired in linear modes with average of 50 shots on a Bruker Daltonics Microflex mass
spectrometer (Bremen, Germany) equipped with a nitrogen UV-Laser operating at 337 nm.
1
Elemental analyses were obtained using a Thermo Finnigan Flash 1112 Instrument. H and
13C NMR spectra were recorded in CDCl
solutions on a Varian 500 MHz spectrometer.
3
Analytical thin layer chromatography (TLC) was performed on silica gel plates (Merck,
Kieselgel 60 Å, 0.25 mm thickness) with F254 indicator. Column chromatography was
5

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performed on silica gel (Merck, Kieselgel 60 Å, 230-400 mesh). Suction column
chromatography was performed on silica gel (Merck, Kieselgel 60 Å, 70-230 mesh).
2
.3. The parameters for fluorescence quantum yields
The fluorescence quantum yield value of the compounds 3 and 4 were determined in
dichloromethane by comparing with the fluorescence of Rhodamine 6G and ZnPc as a
standard, respectively. Fluorescence quantum yields (Φ ) were calculated by the comparative
F
method (Eq. 1) [33].
2
F.AStd.n
ΦF
 ΦF(Std)
(1)
2
Std
FStd.A .n
Where Φ
as the standard for compound 3 (Φ
as the standard (Φ = 0.18 in DMSO) for compound 4 [35]. F and FStd are the areas under the
F
(Std) is the fluorescence quantum yield of standard. Rhodamine 6G was employed
F
= 0.95 in water) [34]. Unsubstituted ZnPc was employed
F
fluorescence emission curve of sample and the standard, respectively. A and AStd are the
2
respective absorbance of the samples and standard at the excitation wavelengths.
η
and
2
Std
η
are the refractive indices of solvents used for the sample and standard, respectively. The
-6
-3
concentration of the solutions at the excitation wavelength fixed at 2x10 mol.dm .
2
.4. Computational Methods
The ground state geometries were created by Density Functional Theory (DFT) [36].
After running an optimization, harmonic vibrational frequencies were computed to test
performed optimization. It was revealed that there were no negative frequencies. The absence
of imaginary frequencies confirmed that the optimization was successfully completed by
chosen method and basis set. In addition to ground state optimization, electron densities of the
targeted compounds were investigated by HOMO-LUMO frontier orbitals theory. All the
computations were performed by Gaussian 09 program [37] on a personal computer using
6

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B3LYP [38] in conjunction with the 6-31G(d,p) and LANL2DZ basis set for anions.
Visualization of the structures was examined by GaussView program [39].
2
.5. Cell lines and cell culture
In this study, human chronic myeloid leukemia cell line (K562) was used. Cells were
cultured in RPMI 1640 medium (Lonza, Switzerland) supplemented with 10% FBS
Biochrom, Germany) and gentamycin (Biological Industries, USA) at 37°C in a humidified
atmosphere with 5% CO
(
2
.
2
.6. Cytotoxicity assay
Cell viabilities were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium
bromide (MTT) assay (Serva Electrophoresis GmBH, Germany) as described previously.
x104 cells/well were seeded into 96-well microplates and incubated for 48 h. Then, cells
1
were washed with phosphate buffered saline (PBS) twice and medium was renewed, and cells
were treated with different concentrations of compounds and incubated for 48 h. Only
mediums including corresponding amount of the compounds were used as blanks, and
acetonitrile, by which compounds were dissolved, treated cells were used as control. Next,
cells were washed with PBS twice, medium was renewed and 10 μl/well MTT solutions (5
o
mg/ml dissolved in PBS) were added onto cells. Cells were incubated at 37 C and 5% CO
2
for 4 h and disrupted by 100 μl/well SDS-HCl solution (10% SDS and 0,01 M HCl)
overnight. Microplates were read at 570 nm by the microplate spectrophotometer (Multiskan
GO; Thermo Fisher Scientific, USA). Optical density values were converted cell viabilities by
using acetonitrile-treated cells as references (100%). Inhibitory concentration 50 (IC50)
referring to the concentration which inhibits half of the cells was calculated from the
logarithmically plotted graph of concentration-cell viabilities for each treatment.
7

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2
.7. Statistical analyses
Cytotoxicity assay was repeated three times as independent experiments with each set
containing quadrate of the same treatment (n=12). Results were analyzed by GraphPad Prism
6
(GraphPad Software, Inc., USA) with Student’s T-Test and significant when p<0.05.
2
.8. Synthesis
Compound 1 [27] and compound 2 [40] were synthesized and purified according to
literature procedure.
2
.8.1. Synthesis of compound 3
A solution of compound 1 (100.0 mg, 0.31 mmol) and compound 2 (45.1 mg, 0.31
mmol) in benzene (30 mL) was treated with piperidine (180 µL) and acetic acid (180 µL)
respectively. The reaction mixture was heated under reflux using Dean-Stark apparatus until
all aldehyde was consumed. The reaction mixture was diluted with water and extracted with
dichloromethane. The combined organic layers washed with brine and dried over sodium
sulphate. The solvent was evaporated, and the product was purified by column
chromatography (SiO
2
, 90%-10% dichloromethane/methanol) to give compound 3 (30.0 mg,
2%). Anal. Calc. for C27 : C, 71.70; H, 5.13; N, 12.39; Found: C, 71.73; H, 5.10; N,
2.35 %. MALDI-TOF m/z Calc. 452.20; found 453.56 [M+H] H NMR (500 MHz, CDCl
8.60 (d, J = 16.80 Hz, 2H), (trans-CH); 7.70-7.45 (m, 5H+4H, Ar-CH); 6.10 (s, 2H); 2,53
2
1
H23BF
2
N
4
+
1
3
)
δ
H
13
(
s, 3H), (CH
3
); 1.97 (s, 6H), (CH
3
) ppm. C NMR (126 MHz, CDCl
3
) δ 167.02, 166.53,
C
1
1
51.69, 151.55, 150.25, 143.84, 137.23, 135.60, 130.58, 128.91, 128.66, 128.49, 128.39,
28.03, 127.94, 127.84, 125.12, 124.90, 124.63, 116.97, 116.57, 109.94, 24.49, 24.33, 18.76
ppm.
2
.8.2. Synthesis of compound 4
A solution of compound 1 (100.0 mg, 0.31 mmol) and compound 2 (90.5 mg, 0.62
mmol) in benzene (30 mL) was treated with piperidine (370 µL) and acetic acid (370 µL)
8

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respectively. The reaction mixture was heated under reflux using Dean-Stark apparatus until
all aldehyde was consumed. The reaction mixture was diluted with water and extracted with
dichloromethane. The combined organic layers washed with brine and dried over sodium
sulphate. The solvent was evaporated, and the product was purified by column
chromatography (SiO , 90%-10% dichloromethane/methanol) to give compound 4 (50 mg,
2
2
1
8%). Anal. Calc. for C35
H
27BF
2
N
6
: C, 72.42; H, 4.69; N, 14.48; Found: C, 72.47; H, 4.65; N,
1
4.43 %. MALDI-TOF m/z Calc. 580.12; found 581.1 [M+H]. H NMR (500 MHz, CDCl
3
)
H
δ 8.11 (d, J = 16.80 Hz, 2H), (trans-CH); 7.75 (d, J = 16.80 Hz, 2H), (trans-CH); 7.68-7.41
13
(m, 5H+8H, Ar-CH), 6.10 (s, 2H), 1.95 (s, 6H), (CH
3
) ppm. C NMR (126 MHz, CDCl
3
) δ
C
1
1
51.89, 151.37, 151.33, 144.81, 143.89, 143.80, 143.71, 143.30, 137.39, 137.15, 136.27,
35.94, 129.74, 129.36, 128.11, 127.99, 127.82, 124.62, 18.70 ppm.
3
. Results and Discussion
3
.1. Synthesis and Structural Characterization
Azaindole known as pyridine-fused heterocyclic compound was employed as a linker
to synthesize monostyryl and distyryl BODIPYs 3 and 4, respectively. The synthesis strategy
began with the formylation of 7-azaindole using hexamethylenetetramine in acetic acid,
which gave the corresponding 7-azaindole-3-carbaldehyde 2 [40]. Knoevenagel reaction
method was utilized for the preparation of the novel BODIPY-azaindole compounds 3 and 4.
The synthesis pathways for preparation of the compounds 3 and 4 are illustrated in Scheme 1.
Accordingly, benzaldehyde (1.0 equiv.) was first treated with 2,4-dimethylpyrrole (2.2 equiv.)
in the presence of TFA in dry DCM, followed by oxidation with p-chloranil at room
temperature. The resulting dipyrrin intermediate was then treated with Et
3
N and BF3.Et O to
2
yield the green fluorescent BODIPY compound 1 [27]. With BODIPY 1 and 7-azaindole
aldehyde 2 in hand, it was of interest to synthesize monostyryl 3 before extending the
9

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Knoevenagel reaction to distyryl. The treatment of the compound 1 with 2 in 1:1 molar ratio
in the presence of acetic acid and piperidine gave monostyryl BODIPY 3 in 22% yield.
Having successfully used Knoevenagel reaction to synthesize monostyryl BODIPY 3,
attention therefore turned to the preparation of the related distyryl BODIPY. For this purpose,
condensation of compound 1 and 2 in 1:2 molar ratio in a Dean-Stark apparatus using
standard conditions produced the compound 4 in 28% yield. All new compounds were
isolated by column chromatography. Identification of the compounds 3 and 4 were performed
1
13
through elemental analysis mass spectrometry, H and C NMR spectroscopy. The results
were consistent with the predicted structures, as shown in the synthesis section. The elemental
analysis results and the mass spectral data for the newly synthesized BODIPY-azaindole
compounds 3 and 4 were consistent with the assigned formulations. The mass spectrum of
compound 3 was obtained by MALDI-TOF MS using 1,8,9-anthracenetriol as the MALDI
matrix, and the spectrum revealed the peak groups representing the protonated molecular ions
at 453.56 Da and molecular ion rupture fluorine at 434.32 Da Figure 1(a). Similarly, the mass
spectrum of compound 4 was showed the protonated molecular ions at 580.308 Da and
molecular ion rupture fluorine at 561.15 Da Figure 1(b). The aromatic and aliphatic protons
1
were observed between 8.60-6.10 ppm and 2.53-1.95 ppm, respectively in the H NMR
spectra. The aromatic carbons for compound 4 and 5 were observed between 167.02-109.94
1
3
ppm and aliphatic carbons 24.49-18.76 ppm in the C NMR spectra.
1
0

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Figure 1 Positive ion and linear mode MALDI TOF-MS spectra of compound 3 (a) and 4 (b).
3
.2. Spectral and Chemosensor Studies
Absorption and fluorescence spectra of azaindole-BODIPY compounds in acetonitrile
8
00
8
00
Compound 3
Compound 3
7
00
7
00
6
00
Compound 4
Compound 4
6
00
5
00
5
00
4
00
4
00
3
00
3
00
2
00
2
00
1
00
1
00
0
0
4
50
500
500
550
550
600
600
Wavelength (nm)
650
650
700
700
750
750
800
800
4
50
Wavelength (nm)
displayed in
Figure 2 exhibit the red-shift with increasing the electron density of the compounds.
While the maximum absorbance wavelength of compounds 3 and 4 were determined at 574
1
1

ACCEPTED MANUSCRIPT
and 655 nm in acetonitrile, the fluorescence emission maxima were found to 595 and 677 nm
8
00
8
00
Compound 3
Compound 3
7
00
7
00
6
00
Compound 4
Compound 4
6
00
5
00
5
00
4
00
4
00
3
00
3
00
2
00
2
00
1
00
1
00
0
0
4
50
500
500
550
550
600
600
Wavelength (nm)
650
650
700
700
750
750
800
800
4
50
Wavelength (nm)
in the same solution, respectively (
Figure 2). The spectral data of azaindole BODIPY compounds 3 and 4 in acetonitrile
were given Table 1. Although it is not the primary issue in this study, the absorption and
fluorescence properties of azaindole substituted BODIPYs 3 and 4 were performed in a
variety of solvent include methanol, ethanol, toluene, acetonitrile, acetone, dichloromethane,
chloroform, dimethylsulfoxide, tetrahydrofuran and benzene at room temperature (Figure S1-
S4). Solvent effect on the absorption spectra was revealed that while the lowest absorption
wavelength was observed in dichloromethane, the highest absorption wavelength was
observed in dimethylsulfoxide for both compounds (Figure S1 and S3). Given the fluorescent
emission spectra, despite the decrease fluorescence emission intensity of the compounds in
the dimethylsulfoxide solvent, red shift was observed in the fluorescence wavelength.
8
00
8
00
Compound 3
Compound 3
7
00
7
00
6
00
Compound 4
Compound 4
6
00
5
00
5
00
4
00
4
00
3
00
3
00
2
00
2
00
1
00
1
00
0
0
4
50
500
500
550
550
600
600
Wavelength (nm)
650
650
700
700
750
750
800
800
4
50
Wavelength (nm)
Figure 2 Excitation (solid lines) and emission spectra (dashed lines) of compound 3 and 4 in
acetonitrile.
1
2

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Table 1 Photophysical properties of azaindole- BODIPYs a
b
c
λ
ab
,
λ
em
,
є ,
∆
(nm)
Stokes
,
K
a
τ
F
d
Detection limit,
(µM)
Compound
Φ
F
4
-1
-1
5
-1
(
nm) (nm)
(10 M cm )
(10 M )
(ns)
4
.173
3
4
574
655
595
677
2.49
21
1.87
0.42
0.14
75.6
CHISQ = 1.285
3
.763
1.28
22
6.13
44.27
CHISQ = 1.752
a
b
c
d
Acetonitrile. Molar extinction coefficients. Lifetime . Fluorescence quantum yield.
The fluorescence lifetime spectra are depicted in Figure 3 for the compound 3 and 4.
The data are collected by using the time correlated single photon counting (TCSPC) technique
in acetonitrile. The lifetimes were found to be 4.17 and 3.76 ns for compounds 3 and 4,
respectively. Furthermore, the fluorescence quantum yields (Φ ) of compounds were
F
calculated 0.42 for compound 3 and 0.14 for compound 4. The fluorescence quantum yield of
compound 3 is higher than that of 4 and this situation is compatible with fluorescence lifetime
results.
Figure 3 Fluorescence decay profile of azaindole-Bodipys (3 and 4) in the presence using
laser excitation source of 390 nm.
The chemosensor properties of the synthesized azaindole-BODIPY 3 and 4 against
−
−
2−, HPO
2−, HSO −
-
some anions aqueous solutions such as I , F , SO
4
4
4
, CH COO were
3
investigated by UV–Vis and fluorescence spectroscopy. All spectral measurements were
performed in acetonitrile solutions. The aqueous solutions of the corresponding sodium
anions were used as the source of anions. To investigate the effect of anions on the absorption
1
3

ACCEPTED MANUSCRIPT
and fluorescence properties of the compounds, the working concentrations of the compound 3
−
−
and 4 were prepared as 3 μM and 10 μM, respectively. 10 μL of 0.1 M different anions (I , F ,
SO 2
−, HPO
2−, HSO −
, CH
COO ) was added to the solution to examine the anion effect on
-
4
4
4
3
the absorption and fluorescence properties of the compounds 3 and 4. It was revealed that no
change in absorption and fluorescence spectra of the titled compounds. Significant changes in
the spectra were observed when bisulfate anion (HSO −
) was added to compounds. Direct
4
comparison of absorption and emission spectra of the compounds are given at Figure 4. Both
compounds exhibit a blue shift (~15 nm) in the absorption spectra (Figure 4(A) and (C)) with
addition of HSO −
anion comparison with other anions. Furthermore, significant decrease was
4
observed for fluorescence intensities of both compounds (Figure 4(B) and (D)). It was clearly
observed that targeted compounds can be found as sensitive "turn-off" fluorescent
chemosensor probes for HSO -
anions. In addition to these results, direct comparison of our
4
results with a recent report by Mahapatra et al. suggest that the position of azaindole moieties
respect to BODIPY core play a significant role for chemosensor properties [32, 41]. It is
worth mentioning while BODIPY with an azaindole group at 8 position is sensitive to
-
-
CH
Figure 4). Our results are consistent with importance of positional effect presents in
literature.
3
COO , in 3 and 5 position of BODIPY do not give any respond to CH COO anion
3
(
1
4

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Figure 4 Absorption and emission spectra of compound 3 (A and B) and 4 (C and D) in 3 μM
in acetonitrile. The data are recorded after addition of 10 μL of 0.1 M different anion solution.
The inserted bar graphs show metal ions selectivity of compounds 3 (upper) and 4 (below).
The purple bars represent the fluorescent intensity of the compounds and the red bars indicate
the fluorescent intensity of the compounds with various ions.
The Continuous Variation method was employed for the determination of the
stoichiometry between the compound 3 and 4 and HSO −
analysis results, the maximum mole fractions for HSO −
compounds. Namely, azaindole-BODIPY compound 3 and 4 and HSO −
anion. According to Job's plot
4
4
anion were observed as 0.5 for both
4
anions preferred
1
:1(L/M) stoichiometry for the formation of complexes between the fluorescent probe and
anion in solution (Figure 5(A) and (C)). As shown in Figure 5(B) and (D), fluorescence
titration experiments with bisulfate anion were performed to obtain more information on the
change in fluorescence properties of azaindole-BODIPY compounds 3 and 4 in the presence
-5
-5
of bisulfate anion (0-125x10 M for compound 3 and 0-65x10 M for comp. 4). Additionally,
the detection limit (DL) and association constant (Ka) were determined based on the
1
5

ACCEPTED MANUSCRIPT
fluorescence titration data [42] (Figure 6). According to the fluorescence titration curve, limits
of detection of bisulfate anion for sensors were calculated as 75.6 and 44.27 μM for
compounds 3 and 4, respectively (Table 1). The association constant (Ka) of azaindole-
−
BODIPY compounds with HSO
4
anion were determined using the Benesi–Hildebrand
equation as follows:
According to this equation, Ka values for compounds (3 and 4) were calculated as 1.87x105
-1
5
-1
M and 6.13x10 M , respectively. The Ka value of compound 4 is higher than that of
compound 3, and consistent with this result, compound 4 has a lower detection limit than 3.In
addition to these photophysical studies, reversibility of the samples was also investigated. The
-
and Compound 4-HSO -
Compound 3-HSO
4
4
solution were treated with 40 μL 0.1 M H
2
O
2
(
Figure S7) [43] This result indicating that the coordination of the sensor with HSO -
4
is
chemically reversible.
1
6

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-
Figure 5 Job’s plots of fluorescence of compound 3 (A) and 4 (C) mixed with HSO
4
anion.
-
Fluorescence titration of compound 3 (B) and (D) with different amount of HSO
4
anion. All
data recorded in 3 μM in acetonitrile. Excitation wavelength is nm.
Figure 6 Calibration curve of fluorescence intensity for compound 3 (A) and 4 (C). Benesi–
Hildebrand Plot of 1/I -I against 1/[ HSO -] for compound 3 (B) and 4 (D). All data recorded
in acetonitrile.
0
4
3
.3. Computational results
The results given in previous section comparison with recent report [32] show a clear
positional effect for azaindole moieties respect to BODIPY core on chemosensor properties
for azaindole-BODIPY compounds. To find out the origin of this effect, we carried out
quantum mechanical calculations to determine the relaxed geometries and their HOMO-
1
7

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LUMO orbitals energies. The all calculations were carried out by means of B3LYP function
with 6-31G(d,p) for both compounds. Firstly, we will focus on optimized geometries for the
compound 3, 4 and modelled structures. Their optimized geometries are given in Figure 7(A).
The dihedral angles between the methyl groups in the BODIPY dye and hydrogens in the
benzene ring are ~ 90° due to the repulsion interaction. After optimization, Electro Static
Potential (ESP) map was created for compound 3 and 4. The ESP maps are given at Figure 8.
The most positive charge is located at around the NH in azaindole indole units. Thus, the
-
HSO
4
anions are interacted with these parts of the compounds (Figure 7(B)) since
anion is 0.5 for both
experimental data indicate that maximum mole fractions for HSO −
4
compounds (Figure 5). The optimizations are completed for three different modelled
structures labeled as model 1, model 2 and model 3 (Figure 7B). Model 1 is for compound 3
-
interacted with HSO
4
anion. Model 2 and model 3 are for compound 4. Model 2 and 3 differ
only the position of anion with azaindole moieties. Model 2 is interacted only one azaindole
moieties and the other model is with both moieties. The calculations are carried out by the
same level of theory with additionally LANL2DZ basis set for anions. The results showed that
the geometric parameters of the modelled structures are slightly changed comparison with
titled compounds (Figure 7(A) and Figure 7(B)) and model 3 is most stable than model 2
(Figure 7B). Thus, model 3 expected to likely lead to experimental data observed here for
-
compound 4 with HSO
4
anion.
1
8

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Figure 7 The optimized structures of (A) compound 3 and compound 4, (B) compound 3 and
−
4
modelled with HSO
4
anion.
Figure 8 The ESP colored maps for compound 3 (left) and compound 4 (right).
Secondly, having the optimized geometries for compounds and modelled structures
given at Figure 7(A) and (B), our attention turned to analyze the HOMO and LUMO orbitals
to complement experimental observations. The orbitals were created by the same level of
theory. The frontier orbitals for compound 3 and 4 are given at Figure 9. The HOMO orbital
1
9

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energies are -4.85 and -4.59 eV for compound 3 and 4, and the LUMOs are -2.38 and -2.44
eV for the same order. Their calculated energy gabs are -2.47 eV and -2.15 eV for compound
3
3
and 4, respectively. The results indicated that compound 4 has smaller gab than compound
. Accordingly, this predicted energy gabs can be explain the result of fluorescence life time
measurements which is compound 4 has shorter fluorescence lifetime than compound 3. Here,
also, it is important to take attention that the position of azaindole moiety affects to electron
distributions. Direct comparison of an azaindole motif placed at the meso (8) position [32] and
3
-position (mono) of BODIPY (compound 3, in this study) showed that HOMO and LUMO
orbitals are located at only BODIPY core for meso position of azaindole moiety and electrons
are more distributed for compound 3 (Figure 9) decreasing with the band gab. This
comparison indicates that perpendicular position of azaindole moiety respect to BODIPY core
causes blocking of electron conjugation. This observation might be the reason for different
selectivity properties of BODIPY core with azaindole moiety for detection of different anions.
Moreover, the HOMO orbital energies for model 1, model 2 and model 3 are -5.94, -6.10 and
-5.83 eV, respectively. The LUMO orbital energies are -3.63, -3.15 and -3.09 eV, with the
same order.
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0

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Figure 9 The frontier orbitals for compound 3 (left) and compound 4 (right).
3
.4. Biological properties
The novel azaindole-BODIPYs 3 and 4 were tested for their cytotoxicity activity since
azaindole derivates have been widely shown to inhibit important kinases [44-46]. The
cytotoxicity of the azaindole-BODIPY conjugates was assessed against K562 cell lines. The
cells were treated with increasing concentrations of compounds 3 and 4. Also, the cells were
treated with these concentrations of compounds 3 and 4 in the presence of 1 μM of NaHSO
to determine whether the treatment of NaHSO was influencing cytotoxicities. The biological
studies revealed that both compound 3 and 4 were able to inhibit cell viabilities efficiently
Figure 10). The novel monostyryl and distyryl BODIPYs displayed cytotoxic activity alone
with IC50 values of 0,621 ± 0,04 and 0,764 ± 0,079 against K562 cell lines, respectively
Table 2). Moreover, the presence of NaHSO in the targeted BODIPY scaffolds, 3 and 4
could not significantly alter the IC50 values; 0,835 ± 0,246 for compound 3 plus NaHSO and
,752 ± 0,076 for compound 4 plus NaHSO (p<0.05). Preliminary biological assays indicate
4
4
(
(
4
4
0
4
2
1

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that BODIPY platforms containing azaindole moiety have a moderate impact on the viability
and proliferation of the K562 cell lines cancer cell line.
Figure 10 Concentration-cell viability correlations of compound 3, compound 3 plus
NaHSO , compound 4 and compound 4 plus NaHSO4.
4
Table 2 IC50 values for related treatments.
Compounds
Compound 3
Compound
IC50 (μM)
0,621 ± 0,04
0
,835 ± 0,246
0,764 ± 0,079
,752 ± 0,076
3
+NaHSO
4
Compound 4
Compound
0
4
+NaHSO
4
2
2

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4
. Conclusion
Two new fluorescent chemosensor candidates for bisulphate anion were successfully
constructed from azaindole and BODIPY platforms. Structures of obtained monostryl and
distryl BODIPYs were clarified and photophysical studies were investigated. According to
spectral results, observations on blue shift in absorbance spectra and meaning decreases in
fluorescent intensities of both compounds indicated that these two hybrids BODIPYs 3 and 4
can sense bisulphate anion. Conducted computational studies showed that compound 3 has
higher gap than compound 4. We conclude that the final Azaindole-BODIPYs can be used as
fluorescent bisulphate anion sensor. A direct comparison of the positionally different placed
of azaindole moiety respect to BODIPY core plays a critical role for selectivity and sensitivity
for anions. Preliminary biological test indicates that the synthesized azaindole substituted
BODIPYs can be valuable candidate as chemotherapeutic agents in cytotoxicity applications.
It is expected that the development of these probes will gain great attention in chemistry,
biology, and medicine science due to the significant harmful effect of bisulphate anion on
human health.
2
3

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Graphical abstract
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8

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Highlights
New azaindole substituted mono and distyryl BODIPYs have been synthesized.► The Fluorescence Sensing
►
properties of the compounds have been studied in acetonitrile► The cytotoxicity of Azaindole-BODIPYs were
examined against living human leukemia K562 cell lines.► Very selective and sensitive fluorescence sensor for
detection of HSO4- anion has been obtained.
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9