Synthesis, characterization and photophysical properties of luminescent non-symmetric 4-pyridyl benzothiadiazole derivatives

Article abstract of DOI:10.1016/j.molstruc.2016.11.050

Two nonsymmetrical π-extended 2,1,3-benzothiadiazoles (BTDs) bearing a 4-pyridyl moiety (BTD-4pyr and BTD-Et4pyr) were synthesized by sequential cross coupling reactions. The structural difference between the two dyes is the presence of a triple bond between the BTD core and the 4-pyridyl moiety in BTD-Et4pyr. The compounds architecture is similar to previously described selective mitochondrial biomarkers. Both compounds exhibit large Stokes shifts (93–137 nm), high fluorescence quantum yields and linear fluorescence response in the nanomolar range. The existence of the triple bond decreased in about 35% the fluorescence measured from BTD-Et4pyr in respect to BTD-4pyr. Solid-state fluorescence of the dyes were measured, producing considerable smaller Stokes shifts than the ones observed in solutions (74 nm for BTD-4pyr and 66 nm for BTD-Et4pyr). X-ray crystallography analysis of BTD-4pyr indicated a quinoid character for the BTD ring and a nonplanar relation between the BTD core and the aryl/heteroaryl groups. DFT calculations pointed out that the LUMO electron density is concentrated over the BTD core. In relation to HOMO, the electron density is distributed mainly at the methoxyphenyl group, in the hydrocarbon fraction of the BTD core and in ethynyl group for BTD-Et4pyr.

Full text of DOI:10.1016/j.molstruc.2016.11.050

Journal of Molecular Structure 1131 (2017) 181e189
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, characterization and photophysical properties of
luminescent non-symmetric 4-pyridyl benzothiadiazole derivatives
a
b
a
b
Alessandra Pazini , Luis Maqueira , Rafael Stieler , Ricardo Q. Auc eꢀ lio ,
Jones Limberger
a
a, b, *
Laboratory of Molecular Catalysis, Institute of Chemistry e UFRGS, Av. Bento Gonçalves 9500, 91501-970, CP 15003, Porto Alegre, RS, Brazil
Chemistry Department, Pontifical Catholic University of Rio de Janeiro, Rua Marqu ^e s de S a~ o Vicente, 225, G aꢀ vea, 22451-900, Rio de Janeiro, RJ, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 September 2016
Received in revised form
Two nonsymmetrical p-extended 2,1,3-benzothiadiazoles (BTDs) bearing a 4-pyridyl moiety (BTD-4pyr
and BTD-Et4pyr) were synthesized by sequential cross coupling reactions. The structural difference
between the two dyes is the presence of a triple bond between the BTD core and the 4-pyridyl moiety in
BTD-Et4pyr. The compounds architecture is similar to previously described selective mitochondrial
biomarkers. Both compounds exhibit large Stokes shifts (93e137 nm), high fluorescence quantum yields
and linear fluorescence response in the nanomolar range. The existence of the triple bond decreased in
about 35% the fluorescence measured from BTD-Et4pyr in respect to BTD-4pyr. Solid-state fluorescence
of the dyes were measured, producing considerable smaller Stokes shifts than the ones observed in
solutions (74 nm for BTD-4pyr and 66 nm for BTD-Et4pyr). X-ray crystallography analysis of BTD-4pyr
indicated a quinoid character for the BTD ring and a nonplanar relation between the BTD core and the
aryl/heteroaryl groups. DFT calculations pointed out that the LUMO electron density is concentrated over
the BTD core. In relation to HOMO, the electron density is distributed mainly at the methoxyphenyl
group, in the hydrocarbon fraction of the BTD core and in ethynyl group for BTD-Et4pyr.
17 November 2016
Accepted 17 November 2016
Available online 18 November 2016
Keywords:
Benzothiadiazole
Fluorescence
Cross-coupling reaction
Optical probe
©
2016 Elsevier B.V. All rights reserved.
1
. Introduction
of interest for both analytical chemistry and chemical biology. In
the analytical point of view, the development of selective and
sensitive dyes increases the range of options to perform tagging,
derivatization and indirect probing for quantitative determinations.
With respect to chemical biology, depending on the structure, the
dyes can be used as bioprobes, capable to selective label cellular
structures/macromolecules, as well as allow the study of dynamic
Many efforts have been made in the design and synthesis of new
-conjugated molecules with luminescent properties. This search
p
is stimulated by the large number of application of these com-
pounds in different areas of light technology, including: OLEDs
constituents [1,2]; luminescent probes for biological [3,4] and
analytical chemistry [5e7]; organic sensitizers for solar cells [8],
among others. In this context, 2,1,3-benzothiadiazole (BTD) de-
cellular processes [3,4]. In this way, some
p-extended benzothia-
diazole (BTD) derivatives have emerged in last years as privileged
scaffolds for selective staining organelles and cellular components.
Representative examples of BTD-based bioprobes are displayed in
Fig. 1.
The green emitters 1a and 1b selectively tagged mitochondria in
three different cell lines. These compounds were designed to
display two intramolecular H-bonds, which are responsible to keep
the planarity e allow a putative ESIPT (excited state intramolecular
proton transfer) as excited state stabilizing process [10]. The dis-
tribution of the fluorescent BTD-based glycoligand 2 was studied in
living and artificial cell system. The compound is able to access the
inner compartment passively and accumulates specifically in
membranes [11]. The compound 3a was produced as a probe for
rivatives with
p-extended conjugation surge as privileged com-
pounds for these applications, thanks to some relevant features,
including: large Stokes shifts, BTD's strong electron withdrawing
ability (which facilitates intramolecular charge transfer stabilizing
processes), relatively high reduction potential and electron affinity
[9].
Concerning the design of novel fluorescent probes, it is a matter
*
Corresponding author. Chemistry Department, Pontifical Catholic University of
Rio de Janeiro, Rua Marqu e^ s de S a~ o Vicente, 225, G aꢀ vea, 22451-900, Rio de Janeiro,
RJ, Brazil.
E-mail address: limberger@puc-rio.br (J. Limberger).
http://dx.doi.org/10.1016/j.molstruc.2016.11.050
0
022-2860/© 2016 Elsevier B.V. All rights reserved.

182
A. Pazini et al. / Journal of Molecular Structure 1131 (2017) 181e189
O
R
R
N
N
N
O
S
S
N
O
R
NH
N
N
N
N
C12H25
C12H25
S
N
N
N
2
N
N
S
3
3
a R = NO2
b R = NH2
S
1a R = H
N
1b R = Br
HO
HO
N
N
X
N
MeO
NH
X
HN
N
NH
5
N
N
S
4
a X = NH
b X = S
4
N
N
S
N
MeO
MeO
N
NH
N
N
N
N
7
S
8
N
S
6
N
N
S
Fig. 1. Representative bioprobes based on BTD core.
hypoxic tumor cells. This NO
however, when it was added to culture of MG63 cancer cells in
hypoxic conditions, a strong red fluorescence was observed. This
2
-BTD derivative is nonfluorescent,
2. Experimental
2.1. Material and instruments
2
emission is due to the reduction of 3a to its fluorescent NH -de-
rivative 3b. Moreover, it was determined that the presence of 3a in
cells stimulates the expression of the nitroredutase, which could
improve the conversion of 3a to 3b [12]. The symmetric BTD de-
rivatives 4 enable high quality images and selective staining for
nuclear dsDNA in human stem cells. The compounds exhibited
large stoke shifts and high stability, representing an interesting
alternative to the commercial imaging probes [13]. The compounds
All solvents (acetonitrile, methanol, acetone, ethyl acetate,
tetrahydrofuran, propan-2-ol, dichloromethane, toluene, pentane
and diethyl ether) were of analytical grade and purchased from
Merck (Germany). Fluorescein sodium salt was form Fluka,
Gemany, and NaOH was from Merck. The monoarylated interme-
diate 3 was synthesized using procedures described in literature
[16]. Cesium fluoride (CsF) and potassium carbonate (K
2
CO
3
) were
ꢀ
5
and 6 were evaluated as mitochondrial marker in cancer cells
dried under vacuum, at 100 C during 1 h before the use. Arilbor-
onic acid/ester were obtained from Sigma-Aldrich and used as
received. All cross-coupling reactions were performed under argon
atmosphere. Toluene, tetrahydrofuran and dioxane were distillated
under sodium/benzophenone before the use in reactions.
[14]. The results were not satisfactory, however it was observed
that the presence of the donator group 4-methoxyphenyl in 5 was
capable to increase the emission intensity and, the presence of the
ethynylpyridyl group of 6 slightly enhanced the mitochondrial
selectivity. Bearing these results in mind, authors synthesized the
bioprobe 7, which possess both 4-methoxyphenyl and ethy-
nylpyridyl groups. This compound proved to be a remarkable
mitochondrial dye for bioimaging experiments, allowing organelle
imaging and revealing the mitochondrial dynamics during the
whole cell division cycle. Another efficient and selective bioprobe
for mitochondrial staining is 8 [15]. This compound structurally
resembles 7, however, lacking the triple bond spacer. Similarly to
observed for 7, the 4-methoxyphenyl group is responsible for the
increasing of the fluorescence intensity whereas the 2-pyridyl
group is related to organelle selection.
Taking into account the outstanding results observed for mito-
chondrial selection observed for 7 and 8, this work describes the
synthesis and the photophysycal characteristics, in solution and in
solid phase, of the fluorescent compounds BTD-4pyr and BTD-
Et4pyr. These dyes were designed considering the structural fea-
tures that ensured intense emission (4-methoxyphenyl group) and
selectivity (pyridyl group) to the previously described BTD-based
bioprobes 7 and 8. The compounds were obtained with good
yields through sequential cross-coupling reactions, display large
Stokes shifts, high fluorescence quantum yields and linear fluo-
rescence response in the nanomolar range. Moreover, the dyes
exhibit absorption/emission spectra and HOMO/LUMO electronic
maps very similar to previous reported dyes.
UVevis absorption spectra were acquired on a Cary 50 single
ꢁ
1
beam spectrophotometer using scan velocity of 1200 nm min
,
spectral bandpass of 10 nm and 1 cm path length quartz cuvettes
(two optically clear faces). Photoluminescence spectra and steady
state photoluminescence measurements were performed on a
PerkinElmer model LS 45 luminescence spectrophotometer with
10 nm excitation and emission spectral bandpass. Scanning was
ꢁ
1
made at 1500 nm min with solutions placed in 1 cm optical
pathlength quartz cuvettes (four optically clear faces). Photo-
luminescence lifetimes were obtained from decay measurements
using a Horiba-Jobin Ivon-IBH time correlated single photon
counting fluorimeter with a 330 nm nanoLED source (N-16, 1.0 ns
nominal pulse duration at 1 MHz repetition rate). Computer pro-
grams supplied by the manufacturer were employed to process the
time resolved data. Nuclear resonance magnetic spectra were
recorded in a Bruker (Germany) model Avance III HD 400 MHZ
spectrometer. Electron spray ionization high-resolution mass
spectrometric (ESI-(þ) HRMS) analyses were performed on a Q-TOF
(Micromass) mass spectrometer (Waters, USA) with the com-
pounds dissolved in a 10% solution of formic acid in acetonitrile. A
Bruker D8 VENTURE diffractometer outfitted with a PHOTON 100
area detector and
¼ 0.71,073 Å) was used to collect the X-ray data for the structural
analysis of BTD-4pyr.
a
graphite monochromator (Mo-Ka,
l

A. Pazini et al. / Journal of Molecular Structure 1131 (2017) 181e189
183
2.2. Synthesis of the BTD-derivatives
Table 1
Crystallographic data and structure refinement parameters for BTD-4pyr.
4
-(4-methoxyphenyl)-7-(pyridin-4-yl)benzo[c] [1,2,5]thiadia-
Molecular formula
C18H13N OS
3
zole (BTD-4pyr): An oven-dried resealable Schlenk flask was
evacuated, back-filled with argon and charged with 4-bromo-7-(4-
methoxyphenyl)benzo[c] [1,2,5]thiadiazole 11 (1.0 mmol, 320 mg),
ꢁ1
Fw [g mol
T [K]
]
319.37
100 (2)
Triclinic
P (ꢁ1)
Crystal system
Space group
a [Å]
3 4
Pd(PPh ) (0.01 mmol, 11.6 mg), 4-Pyridinylboronic acid MIDA ester
ꢁ
1
9.4071 (5)
9.8787(5)
10.0943 (6)
110.942 (2)
116.571 (2)
98.340 (3)
729.49 (7)
2
(
2
2
1.05 mmol, 246 mg), aqueous 1 mol L
K
2
CO
3
solution (2,0 mL,
b [Å]
c [Å]
ꢀ
.0 mmol) and toluene (4 mL). The reaction was stirred at 110 C for
0 h, then allowed to cool-down to room temperature. The mixture
ꢀ
a
b
g
[ ]
ꢀ
was filtered, the solid was washed with toluene (2 ꢂ 5 mL) and the
solution was concentrated under reduced pressure. The crude
material was then purified by column chromatography on silica gel
using hexane/ethyl acetate (10:1), providing 157 mg (49% yield) of
[ ]
ꢀ
[ ]
3
V [Å ]
Z
ꢁ3
r
m
calcd. [g cm
]
1.454
0.230
332
ꢀ
1
ꢁ1
the BTD-4pyr as a yellow solid. Melting point: 203 C (decomp). H
NMR (400 MHz, CDCl
(ppm) 8.82 (d, J ¼ 5.6 Hz, 2H), 8.36 (d,
[mm
]
F (000)
3
) d
Crystal size [mm]
range [ ]
Limiting indices (h, k, l)
0.26 ꢂ 0.24ꢂ 0.07
2.808 to 26.505
ꢁ11 ꢃ h ꢃ 11
ꢁ12 ꢃ k ꢃ 12
ꢁ12 ꢃ l ꢃ 12
18,139
3013 [0.0404]
99.8
3013/0/208
Gaussian
0.95,847 and 0.98,969
0.0350
0.0851
0.0470
0.0921
J ¼ 5.6 Hz, 2H), 8.04 (d, J ¼ 7.4 Hz, 1H), 7.99 (d, J ¼ 8.6 Hz, 2H), 7.85
ꢀ
q
13
(
d, J ¼ 7.4 Hz, 1H), 7.12 (d, J ¼ 8.6 Hz, 2H), 3.93 (s, 3H). C NMR
100 MHz, CDCl 160.15, 154.14, 153.52, 150.15, 144.71, 134.80,
(
3
) d
1
30.55, 129.40, 129.36, 128.89, 126.94, 123.48, 114.18, 77.32, 77.00,
þ
Reflections collected
Reflections unique [Rint
Completeness to max [%]
Data/restraints/param.
Absorption correction
76.68, 55.41. HRMS (m/z) for C18
H
14
N
3
OS (M þ H ): calculated:
]
3
20.0858. Obtained: 320.0844.
q
4
-(4-methoxyphenyl)-7-(2-(pyridin-4-yl)ethynyl)benzo[c]
1,2,5]thiadiazole (BTD-Et4pyr): An oven-dried resealable Schlenk
flask was evacuated, back-filled with argon and charged with 4-
bromo-7-(4-methoxyphenyl)benzo[c] [1,2,5]thiadiazole 11
(0.01 mmol, 7.0 mg), CuI
0.02 mmol, 3.8 mg), 4-ethynylpyridyne (1.10 mmol, 113 mg),
[
Min. and max. transmission
a
R
1
[I > 2
wR [I > 2
(all data)
s(I)]
a
2
s
(I)]
(
1.0 mmol, 320 mg), PdCl
2
(PPh
3
)
2
a
R
1
a
(
wR2 (all data)
S on F2
a
1.044
0.455 and ꢁ0.266
triethylamine (250
m
L) and THF (4 mL). The reaction was stirred at
Largest diff. peak and hole (e.Åꢁ3)
ꢀ
8
0 C for 18 h, then allowed to cool-own to room temperature. The
a
mixture was filtered, the solid was washed with THF (2 ꢂ 5 mL) and
the solution was concentrated under reduced pressure. The crude
material was then purified by column chromatography on silica gel
using hexane/ethyl acetate (8:1) and providing 261 mg (76% yield)
As defined by the SHELXL program [17].
2
.5. Solutions
ꢀ
1
of the BTD-4pyr as a yellow solid. Melting point: 184 C. H NMR
400 MHz, CDCl3)
The solutions of the dyes, used for the solvent effect studies,
were in the concentration of 25
(
d
(ppm) 8.66 (d, J ¼ 6.0 Hz, 2H), 7.95 (d, J ¼ 8.8 Hz,
ꢁ
ꢁ1
1
m
mol L . Fluorescein sodium salt
2
2
d
H), 7.92 (d, J ¼ 7.4 Hz,1H), 7.69 (d, J ¼ 7.4 Hz,1H), 7.58 (d, J ¼ 6.0 Hz,
ꢁ
7
ꢁ6
1
3
solution (3.0 ꢂ 10 e 1.1 ꢂ 10 mol L ) was employed to esti-
H), 7.08 (d, J ¼ 8.8 Hz, 2H), 3.90 (s, 3H). C NMR (100 MHz, CDCl
3
)
mate fluorescence quantum yields. This solution was prepared in
(ppm) 160.3, 155.1, 153.2, 148.7, 135.7, 134.1, 132.2, 130.6, 129.1,
ꢁ1
NaOH aqueous solution (0.1 mol L ) in order to get a standard
solution with fluorescence quantum efficiency ( f) of 0.93, as
indicated by Sj o€ back et al. [22]. Experiments in solid substrate were
made by placing 150 L of the dyes solutions on the center of a
circular Nylon substrate (10 mm diameter) letting it in a desiccator
until total evaporation of the solvent. Lifetime measurements were
1
C
26.5, 126.0, 114.2, 113.5, 92.1, 91.0, 55.4. HRMS (m/Z) for
þ
F
H
20 14
N
3
OS (M þ H ): calculated: 344.0858. Obtained: 344.0871.
m
2.3. X-ray structural determination
ꢁ1
A combination of and scans was carried out to obtain at least
f
u
made using 10
mmol L dyes solutions (dissolved in ethyl acetate).
a unique data set. The crystal structure was solved using direct
methods in the SHELXS program [17]. The final structure was
refined using SHELXL [17], where the remaining atoms were
located from difference Fourier synthesis. Anisotropic displace-
ment parameters were applied to all non-hydrogen atoms, fol-
3
. Results and discussion
The proposed strategy to obtain the target 4-pyrydil-BTD dyes
Scheme 1) begun with the Suzuki cross-coupling reaction between
the 4,7-dibromo-2,1,3-benzothiadiazole and 4-
(
2
lowed by full-matrix least-squares refinement based on F . All
9
hydrogen atoms were placed in ideal positions and refined as riding
atoms with relative isotropic displacement parameters. The crys-
tallographic data and structure refinement parameters are pro-
vided in Table 1.
methoxyphenylboronic acid. This reaction was performed using
conditions described in literature [16] (N,C,P-palladacycle 10,CsF,
dioxane 130 C) providing the monoarylated intermediate 11 (81%
yield in 24 h). In order to obtain the luminescent compound BTD-
ꢀ
4
pyr, the monobrominated intermediate was reacted with the 4-
pyridylboronic acid MIDA ester 12, using another Suzuki protocol,
with a system composed by Pd(PPh , aqueous K CO and toluene.
2.4. Theoretical calculation software
3
)
4
2
3
The structural parameters were estimated using the 6e31G**
basis set [18]. Calculations were carried out by the density func-
tional theory (DFT) method with the Gaussian03 program package
This reaction afforded BTD-4pyr with 49% yield and 40% overall
yield.
For the synthesis of the ethynyl-containing dye BTD-Et4pyr, the
monobrominated intermediate 11 was reacted in a Sonogashira
[
19]. The commonly used B3LYP (Becke, three-parameter, Lee-
eYangeParr) exchange-correlation functional approach [20] was
2 3 2 3
protocol with 13 under classical conditions (PdCl (PPh ) , CuI, NEt
employed as indicated for the study of other organic systems [21].
in THF). This reaction produced the desired product with 76% yield

184
A. Pazini et al. / Journal of Molecular Structure 1131 (2017) 181e189
Cl
MeN
B
O
O
O
O
S
S
S
N
Pd
O
N
N
N
N
N
N
Me
2
P
10 (i-Pr)
N
12
2
MeO
Br
MeO
N
Br
Br
4
-MeOC H B(OH)
6 4 2
Pd(PPh3)4, K CO
PhMe, 110 C, 20h
2
3
CsF, Dioxane
11
81 %
o
BTD-4pyr
9%
9
o
1
30 C, 24h
4
S
N
N
N
13
11
MeO
N
PdCl (PPh ) CuI, NEt3
2
3 2,
o
BTD-Et4pyr
6%
THF, 80 C, 18h
7
Scheme 1. Synthesis of the BTD derivatives BTD-4pyr and BTD-Et4pyr.
and overall yield of 62%.
An expansion of the aromatic region of the H NMR of the dyes is
depicted in Fig. 2. For both compounds, all aromatic/hetero-
parameters are reported in Table 1. Selected bond lengths are listed
in Table 2. The solid-state structure of BTD4-pyr reveals a
nonplanar molecule, with the aryl/pyridyl rings twisting each other
due to the steric hindrance between the hydrogen atoms. The
average dihedral angles between the pyridyl and BTD ring is
1
3
aromatic hydrogens can be observed as doublets, and the
J
H-H
coupling can be easily calculated (the J values are described in
Experimental Section). The chemical shifts of 4-methoxyaryl hy-
drogens H1/H1' and H2/H2' are very similar for both BTD-4pyr and
BTD-Et4pyr respectively appearing around 7.1 ppm and 8.0 ppm.
The insertion of ethynyl spacer between the BTD core and the 4-
pyridyl group shifts upfield both BTD core hydrogens H’BTD in
comparison with HBTD. The triple bond spacer also affects the
chemical shift of the hydrogens H3 (8.36 ppm) and H4 (8.81 ppm)
ꢀ
35.6(2) . This torsion angle is similar to observed for the solid
ꢀ
structures of 4,7-dipyridylbenzothiadiazole (36.8 ) [23]. The tor-
sion angle between the methoxyaryl ring and BTD core of the dye
ꢀ
ꢀ
BTD4-pyr is 46.3(2) , about 15 higher than the angles observed for
the bioprobe 7 [24]. The inspection of the bond distances in the BTD
skeleton suggest a quinoid character for the BTD ring [25,26] since
considerable shortenings of the C8eC9, C10eC11, bonds are
observed (Table 2). The quinoid form is found in many dyes and can
be considered a powerful chromophore that produce intensely
colored compounds [9]. The packing structure of BTD4-pyr is
shown in Fig. 2B. Although the stacking structure is formed, the
interactions between the molecules seems to be weak considering
the molecular plane distances. The distance between the planes of
the BTD rings is 3.33 Å. No short S/N or S/S contacts are observed
in the crystal structures of the compound.
in relation to H3’ (7.58 p.m.) and H4’ (8.66 ppm). In relation to 13
NMR, the dyes spectra are similar (see Supplementary Material),
the major difference is the triple bond carbons of BTD-Et4pyr,
observed at 91.0 and 92.1 ppm.
Single crystals of BTD4-pyr suitable for X-ray diffraction studies
were grown at room temperature from a concentrated solution of
toluene. The molecular structure of the compound is shown in
Fig. 3. The main crystallographic data and structure refinement
C
Fig. 2. Expansion of aromatic region of 1H NMR spectra (400 MHz, CDCl
) for BTD-4pyr and BTD-Et-4pyr.
3

A. Pazini et al. / Journal of Molecular Structure 1131 (2017) 181e189
185
Fig. 3. a) Molecular structure of the dye BTD4-pyr. The ellipsoids are draw with 50% probability level. b) Packing structure of BTD4-pyr.
Table 2
Fluorescence from the dyes were determined in several solvents
and the results are depicted in Fig. 5 and in Table 3. Both com-
pounds showed large Stokes shifts, ranging from 100 to 137 nm for
BTD-4pyr and from 93 to 132 nm for BTD-Et4pyr, which is char-
Selected bond lengths (Å) for BTD4-pyr.
Bond
Bond lenght (Å)
Bond
Bond lenght (Å)
C (8)eC (9)
1.371 (2)
1.420 (2)
1.370 (2)
1.435 (2)
1.434 (2)
1.431 (2)
SeN (1)
SeN (2)
N (1)eC (13)
N (2)eC (12)
1.6102 (14)
1.6141 (14)
1.351 (2)
acteristic of
p-extended benzothiadiazoles [9,32]. These large
C (9)eC (10)
C (10)eC (11)
C (11)eC (12)
C (12)eC (13)
C (13)eC (8)
Dlmax values indicate the high stability of the excited state and a
very efficient intramolecular charge transfer (ICT) in the excited
state, between the methoxyphenyl group (donor) and the BTD ring
1.348 (2)
(acceptor). It is important to mention that this type of donor-
aceptor relation between these groups is considered essential for
the high sensitivity of the BTD mitochondrial markers 7 and 8
The UVevis absorption spectra of the dyes were recorded at
room temperature either in chloroform (Fig. 4a) or in ethyl acetate
[14,15]. For most solvents, BTD-Et4pyr exhibited maximum emis-
sion at slightly higher wavelengths when compared to BTD-4pyr.
This result is in agreement with DFT results (described below),
(
Fig. 4b). In Fig. 4a, two strong absorption bands can be observed for
both compounds. The bands centered at 321 and 311 nm are related
to * [18,27] transitions whereas the absorptions observed at 417
and 418 nm are attributed to intramolecular charge transfer (ICT)
18,27]. In ethyl acetate BTD-4pyr and BTD-Et4pyr presented
since the large
energy gap between molecular excited and ground states.
Fluorescence quantum yields ( ) and the singlet lifetime (
values for both compounds were estimated. BTD-4pyr displayed
p-extension of the ethynyl-BTD dye decreases the
p-p
F
f
f
t )
[
F
f
spectral maximum at 390 and 406 nm, respectively, with FWHM of
about 70 nm. The maximum of absorption observed for BTD-4pyr is
slightly more energetic than observed in literature for the bioprobe
[15], whereas the BTD-Et4pyr absorption is observed in similar
energies than 7 and its 3-pyrydyl analog [14,28]. Estimated molar
of 0.49, about 35% larger than the one BTD-Et4pyr (0.32). Such a
difference can be explained in terms of a more compact and rigid
structure of BTD-4pyr, leading to less efficient non-radiative energy
8
loss. These
F
f
values explain the relative difference in fluorescence
value for BTD-4pyr
9.1 ns) is also larger (about 20%) than the one of BTD-Et4pyr
intensities for these two compounds. The
t
f
absorptivities in ethyl acetate (ε390 and
ε
406
)
were
(
(
[
ꢁ1
ꢁ1
1
9
8,300
L
mol
cm
(log
ε
¼
4.26) for BTD-4pyr and
7.3 ns). As described previously for symmetric BTD derivatives
32], the BTD adjacent triple bond increases the electron-transfer
ꢁ
1
ꢁ1
290 L mol cm (log ε ¼ 3.97) for BTD-Et4pyr. Additionally, the
opt
calculated optical band gaps [29,30] (Egap) were 2.76 eV for BTD-
pyr and 2.68 eV for BTD-Et4pyr, which are in the appropriate
range to be used in organic-light emitting diodes (between 1.5 and
.5 eV) [9,31].
between BTD core and the pyridyl group, consequently
4
decreasing lifetime of BTD-Et4pyr. The radiative decay rate con-
7
ꢁ1
7
ꢁ1
f
stants (F /
t
f
) are 5.4 ꢂ 10 s for BTD-4pyr and 4.4 ꢂ 10 s for
5
ꢁ
1
Fig. 4. Absorption spectra of BTD-4pyr and BTD-Et4pyr in chloroform (a) and in ethyl acetate (b). Concentration of dyes 25 mmol L .

186
A. Pazini et al. / Journal of Molecular Structure 1131 (2017) 181e189
ꢁ
1
Fig. 5. Fluorescence emission spectra of BTD-4pyr and BTD-Et4pyr in different solvents. Concentration the dyes: 10 mmol L .
Table 3
Fluorescence excitation and emission maxima and Stokes shift of BTD-4pyr and BTD-Et4pyr in different solvents.
lmaxexc (nm)
Polarity index
Dielectric constant
E
N
Dlmax
Solvent
BTD-4pyr
BTD-Et4pyr
BTD-4pyr
BTD-Et4pyr
Acetonitrile
Acetone
Methanol
Ethyl Acetate
THF
Propan-2-ol
2 2
CH Cl
Toluene
386
390
414
392
396
396
415
400
408
408
410
409
411
410
412
414
5.8
5.1
5.1
4.4
4.0
3.9
3.1
2.4
37.5
2.07
32.7
6,02
7.58
17.9
8.93
2.38
0.460
0.355
0.762
0.228
0.605
0.546
0.309
0.099
137
128
131
120
118
122
115
100
119
113
132
107
109
120
109
93
BTD-Et4pyr.
expected). In Fig. 3, it can be seen that the polarity of solvent (the
effect of four solvents are shown) has a higher influence on the
intensity and spectral positon of BTD-4pyr emission band than for
BTD-Et4pyr.
Upon illumination by a hold-hand UV lamp, both dyes are bright
emissive in the green. In order to quantify this effect, the fluores-
cence spectra of the compounds were obtained directly from the
solid materials (onto a Nylon substrate). Broad spectra were ob-
tained with significantly smaller Stokes shifts, in comparison with
The more polar the molecule, the stronger the effect of more
polar solvents on the energy relaxation processes that, in turn,
results in the increasing of the observed Stokes shift (Dlmax). Taking
into consideration the position of the fluorescence excitation band
of the dyes (in terms lexc), it cannot be seen a clear direction for the
solvatochromism using different criteria to establish the polarity of
the solvent (polarity index, dielectric constant or E TN (30)). However,
for BTD-4pyr it can be clearly seen an increasing of Dlmax as the
polarity index increases, but this is not so clear for BTD-Et4pyr
solution. For BTD-4pyr the
Dlmax ¼ 74 nm) while for BTD-Et4pyr the
483 nm (Dlmax ¼ 66 nm) as can be seen in Fig. 7.
l
exc
/
l
em pair was 406/480 nm
(
Table 3). In contrast, for BTD-Et4pyr, a clear tendency for the
(
exc
l /l
em pair was 417/
increasing of Dlmax was observed as the E TN (30) value increased. Such
results are an indication of the stabilization of the luminophore
excited state relative to the ground state as the solvent polarity
increased and that these dyes permanent dipole moment increases
upon excitation.
In order to better understand the relationship between the
structure of the dyes and its electronic properties, it was performed
a quantum mechanical theoretical investigation. Self-consistent
density-functional calculations (DFT) were used to provide accu-
rate description of the electronic distribution and atomic arrange-
ments of these compounds and a qualitative estimate of their
HOMO and LUMO energies. The molecular structures were opti-
mized considering a vacuum environment, using Gaussian03
package [19].
The Lippert-Mataga [33] plot (Fig. 6), which establish the rela-
tionship between the Stokes shift (cm ) and solvent polarity
ꢁ
1
N
values (reported in terms of E
T
) [34] expecting that the higher the
N
E
T
the wider the Stokes shift (expected order of Spectral shifts:
Methanol
ol acetonitrile
>
THF
acetone
>
propan-2-
ethyl
>
>
>
dichloromethane
>
In Fig. 8, the ground-state structure parameters of the BTDs and
the contours of their HOMO and LUMO are shown. As can be seen,
for both compounds, the LUMO electron density is concentrated
over the BTD core. In relation to HOMO, the electron density are
distributed mainly at the methoxyphenyl group (donor), in the
hydrocarbon fraction of the BTD core and in ethynyl group for BTD-
Et4pyr.The LUMO and HOMO distribution orbital corroborates the
highly efficient donor-acceptor character of these dyes, with
intramolecular charge transfer (ICT) between BTD core (electron
acceptor group) and the methoxyphenyl (donor group). The
acetate > toluene). An almost perfect correlation between solvent
polarity and Stokes shift was found for BTD-Et4pyr (R > 0.80),
with only THF producing a magnitude of the spectral shift less than
what was expected. The presence of the triple bond might facilitate
solvation thus producing the more stable excited states in the
presence of more polar solvents. For BTD-4pyr the correlation does
not exactly obey the polarity order (R > 0.44) but still shows higher
spectral shifts for the relatively more polar solvents (exception
made for THF that also produced a smaller Stokes shift than
2
2

A. Pazini et al. / Journal of Molecular Structure 1131 (2017) 181e189
187
Fig. 6. Stoke's shift (cmꢁ1) of dyes in function of EN
values for in different solvents. BTD-4pyr (R >0.80), BTD-Et4pyr (R >0.44). Concentration of dyes: 10
2
2
m
ꢁ1
mol L .
T
HOMO-LUMO band-gap energies were also calculated and the re-
sults are shown in Table 4. The obtained values were 3.15 eV for
BTD-4pyr and 2.95 eV for BTD-Et4pyr. It is noteworthy that the
electronic map of the fluorophores is very similar to previously
described bioprobes and the band gap of the 4-pyridyl fluorophores
are smaller than the observed for 2-pyridyl analogs 7 and 8 [14,15].
In terms of optimized conformation, the calculated structure of
the BTD-Et4pyr showed a quasi-coplanar conformation between
pyridyl ring and the BTD core, whereas the BTD-4pyr presented a
twist of the pyridyl group. The torsion angle calculated between
ꢀ
pyridyl groups and BTD core (35.1 ) was similar to those obtained
ꢀ
from X-ray diffraction analysis (35.6 ). In general, the dihedral
angles and energies values calculated are similar to others BTD-aryl
and BTD-ethynylaryl derivatives already reported [14,27]. With
respect to bond distances, the DFT results and X-ray diffraction of
BTD-4pyr are in agreement, since both techniques indicated a
shortening in C8eC9 and C10eC11 bonds, suggesting a quinoid
character for the BTD skeleton. A similar shortening was also
observed in DFT results for BTD-Et4pyr (Table S3 of Supplementary
Material).
Fig. 7. Fluorescence excitation and emission spectra of the dyes in the solid state of
BTD-4pyr and BTD-Et4pyr.
Fig. 8. Optimized structures and frontier molecular orbitals of BTD-4pyr and BTD-Et4pyr, calculated in the gas phase.
Table 4
Theoretical calculated data for BTDs synthetized at B3LYP/6-31G** in gas phase.
BTD
HOMO (eV)
LUMO (eV)
HOMO-LUMO gap (eV)
D.A (degree) (BTD/Pyridyl)
D.A (degree) (BTD/4-MeOPh)
BTD-4pyr
BTD-Et4pyr
ꢁ5.70
ꢁ5.60
ꢁ2.55
ꢁ2.65
3.15
2.95
35,1
4.9
35,3
34.0

188
A. Pazini et al. / Journal of Molecular Structure 1131 (2017) 181e189
Fluorescence response of both compounds were studied using
analytical curves prepared in ethyl acetate within the nmol L
Acknowledgements
ꢁ1
range and signal measurements made at the maximum excitation/
emission wavelengths (Fig. 9). The analytical response was linear in
J.L. thank CNPq and VRAC-PUC-Rio for financial support. A.P. also
thank CNPq for scholarship. R.Q.A. thank CNPq and FAPERJ for
scholarships. We are grateful to Prof. Sonia Louro for the fluores-
cence lifetimes experiments and to Prof. Jairton Dupont for the use
of LAMOCA facilities.
ꢁ1
2
the range covering 0.1e100 nmol L with R of 0.997 for BTD-4pyr
and 0.999 for BTD-Et4pyr. The linear equation models were: FBTD-
9
8
4
pyr ¼ (7.85 ꢂ 10 ± 1.5 ꢂ 10 )CBTD-4pyr þ (14.28 ± 6.1) and FBTD-
9 7
Et4pyr ¼ (4.40 ꢂ 10 ± 1.6 ꢂ 10 )CBTD-Et4pyr þ (4.54 ± 0.7), where F is
measured fluorescence intensity and C is dye concentration. From
these curves, the limits of detection were calculated (using the 3
Appendix A. Supplementary data
s
blank/m, where sblank was the standard deviation of the blank and m
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.molstruc.2016.11.050.
is the sensitivity of the analytical curve) and the values were
0
ꢁ1
ꢁ1
.17 nmol L for BTD-4pyr and 0.27 nmol L for BTD-Et4pyr.
These results indicates the good quality of the optical response of
these dyes in potential quantitative applications as analytical
probes or markers.
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