On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes

Article abstract of DOI:10.1016/j.bmcl.2010.08.073

Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolecular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent monolayer of human stem-cells (tissue). All tes

Full text of DOI:10.1016/j.bmcl.2010.08.073

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6001–6007
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
On the use of 2,1,3-benzothiadiazole derivatives as selective live cell
fluorescence imaging probes
Felipe F. D. Oliveira a,b, Diego C. B. D. Santos a,b, Alexandre A. M. Lapis c, José R. Corrêa a,b
Alexandre F. Gomes d, Fabio C. Gozzo d, Paulo F. Moreira Jr. e, Virgínia C. de Oliveira a,b, Frank H. Quina b,e
,
,
Brenno A. D. Neto a,b,
⇑
a Laboratory of Medicinal and Technological Chemistry, University of Brasília (IQ-UnB), Campus Universitário Darcy Ribeiro CEP 70904-970, PO Box 4478, Brasília, DF, Brazil
b National Institute of Catalysis (INCT-Catalysis), Brazil
c Universidade Federal do Pampa, Unipampa/Urcamp, Bagé, RS, Brazil
d Institute of Chemistry, State University of Campinas, Brazil
e Institute of Chemistry, USP, São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolec-
ular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent
monolayer of human stem-cells (tissue). All tested dyes were compared with the commercially available
DAPI and gave far better results.
Received 6 August 2010
Revised 12 August 2010
Accepted 16 August 2010
Available online 20 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Benzothiadiazole
ESIPT
Live cell-imaging
Stem-cells
The development of new and efficient fluorophores to detect,
quantify or to observe cellular process is currently of a great inter-
est and has been recently reviewed.1 The design and synthesis of
new fluorophores that display any induced fluorescence signal
change (e.g., intensity and/or emission wavelength or shape) when
a fluorophore-containing probe specifically binds to a target is par-
ticularly desirable.2 Ultrafast excited state intramolecular proton
transfer (ESIPT) processes greatly increase the stability of a mole-
cule in the excited state, avoiding undesirable photochemical reac-
tions.3 Additionally, it has been demonstrated that the ESIPT
process is microenvironmentally sensitive; thus, when the emis-
sion is characteristics of an ESIPT compound is dependent on its
microenvironment, its fluorescence emission are expected to
change under different conditions.4 Indeed, molecules showing
ESIPT fluorescence have been employed as probes of microenviron-
ments in biological systems.5 Molecules that contain two sites
capable of undergoing ESIPT processes, a molecular architecture
that greatly increases the molecule’s stability in the excited state,
have also been reported.6
In previous works, we showed that the 2,1,3-benzothiadiazole
(BTD)7 core and its derivatives are potential candidates for use in
organoelectronic devices,8 as molecular fluorescent probes for
selective dsDNA detection9a and that they are efficiently used in
real-time PCR.9b In the present work, we designed and synthesized
new 2,1,3-benzothiadiazole derivatives having four sites poten-
tially capable of participating in ESIPT processes and report photo-
physical and electrochemical data and preliminary studies of
application in live cell-imaging in a selective staining of dsDNA
in human stem-cells.
The dyes were synthesized in good yields as shown in Scheme 1
and were fully characterized (see Supplementary data). The known
ESIPT fluorophores BI and BT were synthesized using a previously
described methodology by Campo et al.10 The new derivatives,
BTDBI and BTDBT, were synthesized by Buchwald-Hartwig amina-
tion of 4,7-dibromo-2,1,3-benzothiadiazole (3), employing an
adaptation of
a procedure recently described by Hirao and
coworkers.11
In the Supplementary data, we describe the general ESIPT pro-
cess (see Scheme S1). As indicated in Scheme 2, the molecular
architecture of the dyes BTDBI and BTDBT has been designed to
permit, in principle, any one of four different ESIPT processes. In
two of the four possible ESIPT processes, intramolecular proton
⇑
Corresponding author. Tel.: +55 61 31073867; fax: +55 61 32734149.
E-mail address: brenno.ipi@gmail.com (B.A.D. Neto).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.073

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F. F. D. Oliveira et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6001–6007
HO
HO
NH2
Y
N
HO2C
PPA
+
X
1a Y = NH2
1b Y = SH
NH2
NH2
BI (X = NH)
BT (X = S)
2
HO
OH
N
N
X
Br
Br
N
N
X
S
3
N
N
DPEPhos
Pd(OAc)2
H
H
N
N
Et3N
S
Ph Me
BTDBI (X = NH)
BTDBT (X = S)
Scheme 1. Synthesis of the dyes BI, BT, BTDBI and BTDBT.
ESIPT
process
(possibility 4)
ESIPT
process
(possibility 1)
H
O
O
H
N
X
N
X
N
N
H
H
ESIPT
ESIPT
N
N
process
process
S
(possibility 3)
(possibility 2)
BTDBI (X = NH)
BTDBT (X = S)
Scheme 2. Four possibilities for ESIPT process to undergo in the excited state of the fluorophores BTDBI and BTDBT.
transfer would occur via a six-membered ring (possibilities 1 and
4) and, in the other two, via a five-membered ring (possibilities 2
and 3). Both five- and six-membered ring ESIPT processes have
been studied in other systems, where they efficiently stabilize
the molecules in the excited state.5,12 Moreover, upon intercala-
tion, one side of the symmetrical dye would be able to allow the
molecule to undergo ESIPT process in two different sites.
Differential scanning calorimetry (DSC) was used to determine
the thermal properties of all four compounds (melting or decom-
position temperatures). Photophysical properties of all compounds
were also investigated and are summarized in Table 1.
tween the BTD unit and the substituent at positions 4 and 7.14 It
is well known that molecules that undergo ESIPT processes display
a large Stoke shift. BI, BT, BTDBI and BTDBT exhibit large molar
extinction coefficients (e) in acetonitrile (log e = 3.90, 3.72, 3.91
and 3.93, respectively, for 5.00 Â 10À5 M solutions). BI and BT are
almost insoluble in water, but the use of ultrasound permits the
preparation of highly dilute solutions for which no precipitation
is noted after standing for a few minutes. The derivatives BTDBI
and BTDBT showed higher solubility in water. The strategy of com-
bining the advantages of a BTD15 core with ESIPT processes thus
appears to be a good approach for improving the photophysical
properties of the new compounds.
The large observed Stokes shift is a consequence of the high sta-
bility in the excited state and the ESIPT process. Moreover, such a
large Stokes shift and solvent-dependent emission indicates effec-
tive intramolecular charge transfer (ICT) in the excited state be-
Spectrophotometric titrations: All dyes were assayed by spectro-
photometric titration against dsDNA (calf thymus) or the BSA pro-
tein (bovine serum albumin). Water (Milli-Q purified) was used as
Table 1
UV-vis and fluorescence data for BI, BT, BTDBI and BTDBT
Compd
kmax (abs)a
kmax (em)a
Stokes shifta
Uf
BI
BT
BTDBI
BTDBT
346 (344),b 361 (362),b 338
371 (369),b 389 (389),b 359
348, 356, 353
478, (479),b 425, (423),b 456
513, (508),b 478, (477),b 462
444, 438, 462
132, 64, 127
142, 89, 103
96, 82, 109
101, 95, 118
0.022
0.0046
0.016
0.016
344, 345, 345
445, 440, 463
a
EtOH, MeCN, H2O, respectively.
Literature.13
b

F. F. D. Oliveira et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6001–6007
6003
medium for these experiments as a prelude to future cellular
experiments (live cell-imaging). The presence of phosphate buffer
(pH 7.0) did not significantly alter the results obtained. Suppos-
edly, the binding with the biomolecule would result in a different
spectrum during the titration. The use of very dilute solutions of
the dyes (1.0 Â 10À5 M) failed to reveal significant changes in the
spectra (see Figs. S1 and S2 in the Supplementary data). However,
changes, as expected, but the intrinsic fluorescence remains un-
changed due to the large -extension. Moreover, the large exten-
sion of the -conjugation turns both, BTDBI and BTDBT, highly
stable. Despite the pH value, that could lead both BTD-derivatives
to anionic or cationic species, almost no absorption maximum shift
is noted. It is due to the fact that the charge (positive or negative) is
p
p
very well stabilized in the highly extended p-systems. A reference
with solutions of a much higher concentration (50
l
M),16 better re-
work has been published20 demonstrating that in aqueous solu-
tions at near neutral pH values species have a tendency to be pre-
dominantly neutral, whereas at lower and higher pH values they
tend to be cationic and anionic, respectively. Nevertheless, the fact
that BTDBI and BTDBT have the highest fluorescence in the range
of physiological pH values makes these fluorophores excellent can-
didates for biological applications such as live cell-imaging.
It is noted that for BTDBI and BTDBT, the pH-induced changes
in fluorescence intensity are reversible, indicating that protonation
and deprotonation are reversible and no decomposition is noted.
This was not the case for BI and BT, which probably suffer some
degradation in very acidic or basic solutions.
sults were obtained. Figure 1 shows representative results for the
dye BTDBT. Results for all of the fluorophores are provided in the
Supplementary data (Figs. S3–S6).
The use of benzoxazole,17a benzothiazole17b and benzoimidaz-
ole17c derivatives for protein detection has been described, so spec-
tral changes for our compounds could be expected in the presence
of BSA protein. Indeed, spectrophotometric titrations gave better
results for protein detection than for the nucleic acid under the
conditions studied.
Spectrofluorimetric titrations: Spectrofluorimetric detection is far
better when compared to spectrophotometric detection. Indeed,
spectrofluorimetric titrations gave very interesting results even
at low dye concentrations. The best results for protein and nucleic
acid detection were obtained using BTDBT (Fig. 2; see Figs. S7–S10
in the Supplementary data for the data for all dyes). However, for
protein detection BTDBT was not as good as expected.
Electrochemical analysis: Since protein detection was not very
effective, and the selectivity was very good for dsDNA, we decided
to limit our study of electrochemical methods to DNA detection.
DNA interactions were studied by using cyclic voltammetric anal-
ysis. The cyclic voltammograms (CV) are shown in Figure 4.
Larger electrochemical windows were not tested in order to
avoid any solvent oxidation and/or reduction. In all cases, we ob-
served multielectron irreversible reduction steps during cathodic
potential sweeps in the presence of DNA or for pure DNA itself.
No cathodic reduction processes were observed for any of the pure
fluorophores in the same electrochemical window. However, the
anodic sweep for the dyes BI and BT provided very interesting re-
sults. BI showed an anodic peak at 0.48 V associated with the peak
at 0.38 V in a reversible charge transfer process. Upon DNA addi-
tion, a shift of these peaks to 0.65 and 0.27 V is observed and the
charge transfer is quasi-reversible. BT also showed an anodic peak
at 0.70 V associated with the peak at 0.53 V in a reversible charge
transfer process. When the DNA is added to the system, the peak at
0.70 V is shifted to 0.57 V and the oxidation process become irre-
versible, since no peak is noted during the reverse sweep. In con-
trast, neither BTDBI nor BTDBT showed any oxidation or
reduction processes in the electrochemical window studied, clearly
indicating their high electrochemical stability. Upon DNA addition,
In contrast, BTDBI gave rather poor results (Fig. S9 in the Sup-
plementary data), most probably due to its high hydrophilicity.
This lowers the affinity of the dye for the biomolecules and results
in only small spectral changes, making it less than desirable as a
probe for the spectrofluorimetric detection and quantification of
biomolecules. Although BI and BT gave reasonable results, their
poor solubility means that they are not very adequate for detecting
biomolecules. Based on these considerations, BTDBT (Fig. 2) is
clearly the best fluorophore among the four tested in this work.
pH dependence of the fluorescence: The pH dependence of ESIPT
processes is well known and the species involved can be neutral,
anionic or cationic, depending on the pH range.18 For instance,
we have recently shown this pH dependence for the substrate of
IGPS enzyme from Mycobacterium tuberculosis bacillus and its
importance in tryptophan biosynthesis.19 In this work, very differ-
ent behaviors were observed for the four dyes (Fig. 3). While the
fluorescence spectra of BI and BT are strongly dependent on the
solution pH (shape, intensity and absorption maximum shift), only
the fluorescence intensity of BTDBI and BTDBT changes, with the
shape of the spectra being almost unchanged. While BI and BT
are strong dependent on the pH value (shape, intensity and absorp-
there was a pronounced change in the current (lA), especially for
BTDBT, consistent with its high affinity for dsDNA. In some re-
cently published works,21 a similar behavior could be noted upon
dsDNA addition to a small molecule-containing solution. The
substantial change in the peak current can be attributed to the
tion maximum shift), the large
p-extension of BTDBI and BTDBT
makes the shape of the spectra almost unchanged. The intensity
1.6
dsDNA concentration (0-300 μM)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
Protein concentration (0-1000 μg/mL)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
50 μM (dye concentration)
50 μM (dye concentration)
0.0
200
250
300
350
λ (nm)
400
450
250
300
350
λ (nm)
400
450
Figure 1. Typical spectral behavior for the spectrophotometric titration of BTDBT with dsDNA (left) or BSA (right).

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F. F. D. Oliveira et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6001–6007
R2 = 0.98
dsDNAconcentration (0-300μM)
250
200
150
100
50
100
95
90
85
80
75
10 μM (dye concentration)
0
400
425
450
475
500
λ (nm)
525
550
575
600
0
50
100
150
DNA (μM)
200
250
300
300
250
200
150
100
50
R2 = 0.99
Protein concentration (0-1000 μg/mL)
100
96
92
88
84
10 μM (dye concentration)
0
400
450
500
550
600
0
200
400
600
800
1000
λ (nm)
Enzyme concentration (μg/mL)
Figure 2. Representative spectrofluorimetric titration of BTDBT with dsDNA (upper) or BSA (lower).
pH= 4.5
pH= 3.5
pH= 6.5
pH= 7.0
BT
BI
100
80
60
40
20
0
100
80
60
40
20
0
pH= 7.0
pH= 7.5
pH= 6.5
pH= 1.5
pH= 1.0
pH= 0.5
pH= 12.5
pH= 1.5
pH= 7.5
pH= 13.0
pH= 13.5
pH= 12.5
pH= 13.0
pH= 13.5
pH= 1.0
pH= 0.5
400
450
500
λ (nm)
550
600
400
450
500
λ (nm)
550
600
pH= 7.0
pH= 6.5
pH= 7.0
BTDBT
BTDBI
100
80
60
40
20
0
100
80
60
40
20
0
pH= 7.5
pH= 6.5
pH= 7.5
pH = 12.5
pH= 12.5
pH= 13.0
pH= 13.5
pH = 13.0, 13.5, 1.5
pH= 1.5
pH= 1.0
pH= 0.5
pH= 1.0
pH= 0.5
400
450
500
λ (nm)
550
600
400
450
500
λ (nm)
550
600
Figure 3. Dependence of the probe fluorescence on pH in aqueous solutions. See Supplementary data (Figs. S11–S12) for the spectra at all of the pH values tested for the four
dyes.

F. F. D. Oliveira et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6001–6007
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200
100
0
200
100
0
-100
-200
-100
-200
-300
-400
-300
BI
BT
BT + DNA
DNA
BI + DNA
-400
DNA
-1.0
-0.5
0.0
0.5
1.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
E / V (SCE)
E / V (SCE)
200
100
0
200
100
0
-100
-200
-300
-400
-100
-200
-300
-400
BTDBT
BTDBT + DNA
DNA
BTDBI
BTDBI + DNA
DNA
-1.0
-0.5
0.0
0.5
1.0
-1.0
-0.5
0.0
0.5
1.0
E / V (SCE)
E / V (SCE)
Figure 4. Cyclic voltamograms for all four dyes (1.00 mM). In MeCN (0.10 M TBAPF6) recorded at a scan rate of 300 mV/s. The dsDNA concentration was 300
Note the large difference in the current ( A) for BTDBT between the biomolecule (DNA) alone and for DNA + BTDBT.
l
M in all cases.
l
Figure 5. Live cell-imaging using DAPI (commercially available) and adhered human stem-cells. Note in this picture that DAPI is not as selective as the designed probes and is
also visualized in the cellular membrane and in other cellular components (right picture). Phase contrast (left) and fluorescence profile (right).
decrease in free fluorophore concentration due to the formation of
a diffusing, heavy molecular weight dye-DNA adduct. We recently
showed the high affinity of BTD-containing intercalators and
dsDNA.9b Peak potential shift could be attributed to the intercala-
tion of a planar and rigid moiety of the molecule into the stacked
base pairs domain of dsDNA, as discussed elsewhere.22
Live cell-imaging analysis: Live cell-imaging experiments were
performed using human stem-cells and murine macrophage cells
(see Experimental details in the Supplementary data). In these
experiments, all dyes were tested with both suspended and ad-
hered cells. Initially, suspended cells (macrophage cells) were used
for testing the viability of the use of the dyes. The results showed
that dsDNA was preferentially stained in all cases (see Figs. S13–
S16 in the Supplementary data). Moreover, a more complex cellu-
lar model consisting of a confluent monolayer of human stem-cells
(tissue) was then tested. All four dyes were capable of transposing
the cellular membrane and successfully and selectively stained
dsDNA inside the human stem-cells (see Fig. 5 and Figs. S17–S21
of the Supplementary data for full-size images).
It is important to highlight the importance of a selective dsDNA
staining. The high selectivity allows the study of any nucleic acid
process without interfering in any enzyme or protein related to a
specific process.
Despite the potentially quite useable staining result, BI and BT
precipitated during the experiment due to their very low solubility
in aqueous media, making them less than appropriate for this kind
of experiment, especially because they can precipitate in the intra-
cellular media.

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F. F. D. Oliveira et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6001–6007
Figure 6. Live cell-imaging of adhered human stem-cells using all four dyes and DAPI (commercially available). Phase contrast (left) and fluorescence micrographs (right).
From top to bottom, the dyes are: BI, BT, BTDBI, BTDBT and DAPI. Note that BTDBT gave the most efficient dsDNA staining.

F. F. D. Oliveira et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6001–6007
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The designed fluorophores BTDBI and BTDBT, however, proved
References and notes
to be exceptional for this purpose, due in large part to their high
dsDNA affinity and their chemical and photochemical stability,
mainly BTDBT. The cellular experiments leave no doubt about
the high affinity of the new dyes for dsDNA. The commercially
available dye DAPI, which is among the most widely used molec-
ular probe for dsDNA staining, was tested in the same confluent
monolayer of human stem-cells in order to compare the results
with those for our molecular probes. It is known that DAPI com-
monly associates with other cellular components in addition to
dsDNA, since its selectivity is not as good as expected to be. For in-
stance, DAPI adsorbs to the cellular membrane resulting in a
strong background signal even at relatively high dilutions (Fig. 5
and Fig. S22).
In the case of both BTDBI and BTDBT (Fig. 6), the images are
quite clear, with little or no background, indicating that no signif-
icant amount of these two fluorophores may remain adsorbed in
the membrane. Actually, they are capable of transposing the mem-
brane without adhering to it. Although both designed dyes are
much better than DAPI, comparison of the results for BTDBI and
BTDBT indicates that BTDBT gave the best overall results as a
live-cell dsDNA stain.
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CAPES, CNPq, FINEP, Finatec, FAPESP and FAPDF are acknowl-
edged for partial financial support. BAD Neto is in debt with Prof.
Sônia M. Freitas and Prof. Paulo A.Z. Suares. B.A.D. Neto, F.C. Gozzo
and F.H. Quina also thank CNPq for research fellowships.
Supplementary data
Supplementary data (spectral data) associated with this article
can be found, in the online version, at doi:10.1016/j.bmcl.2010.
08.073.