New sensitive fluorophores for selective DNA detection

Article abstract of DOI:10.1021/ol701708y

4,7-Disubstituted benzothiadiazoles containing 1-arylethynyl and 4-methoxyphenyl groups are selective photoluminescent "light up" probes to duplex DNA with unprecedented sensibility in both spectrophotometric and spectrofluorimetric measurements.

Full text of DOI:10.1021/ol701708y

ORGANIC
LETTERS
2007
Vol. 9, No. 20
4001-4004
New Sensitive Fluorophores for
Selective DNA Detection
Brenno A. D. Neto,*,† Alexandre A. M. Lapis,‡ Fabiana S. Mancilha,‡
Igor B. Vasconcelos,† Caroline Thum,† Luiz A. Basso,† Dio´genes S. Santos,† and
Ja1
ırton Dupont*,‡
Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF),
Tecnopuc, PUC-RS, Brazil, and Institute of Chemistry, UFRGS Porto Alegre,
RS 90501-970, Brazil
brenno.ipi@gmail.com; dupont@iq.ufrgs.br
Received July 18, 2007
ABSTRACT
4,7-Disubstituted benzothiadiazoles containing 1-arylethynyl and 4-methoxyphenyl groups are selective photoluminescent “light up” probes to
duplex DNA with unprecedented sensibility in both spectrophotometric and spectrofluorimetric measurements.
Biosensor technologies that focus on the direct detection of
nucleic acids are currently an area of tremendous interest as
they play a major role in clinical, forensic, and pharmaceuti-
cal applications.1,2 The molecular probes that cause an
increase in both absorbance and emission intensity (“light
up” probes) by association with the host biomacromolecules
(e.g., DNA, RNA, and proteins) are very useful photolumi-
nescent markers in genomics and proteomics.3 These simple
and straightforward spectroscopic methods are especially
advantageous because small organic dyes absorb and emit
at wavelengths that do not interfere with the absorption of
the DNA bases (λ max ≈ 260 nm). Indeed, spectrophoto-
metric and spectrofluorimetric titrations are direct method-
ologies that indicate the association of a specific dye with
DNA.4
The ability of planar polycyclic aromatic molecules to
intercalate, i.e., to be inserted between two consecutive base
pairs of DNA, is the basic molecular geometry of many
intercalators that are used in antitumor chemotherapy,5 as
DNA cleavage agents6 and fluorescent DNA intercalators,7
and for various other purposes.8 However, in the case of
small molecular organic fluorophores, this association is not
very clear, mainly because of the great diversity of the
possible resulting structures.9 Cationic organic dyes normally
enhance the propensity of a small probe to bind to DNA,
most via an interaction of the positive charge with the
phosphate backbone in the double-strand DNA macromol-
ecules.10,11 However, these charged dyes have some draw-
(5) (a) Denny, W. A.; Baguley, B. In Molecular Aspects of Anti-Cancer
Drug-DNA Interaction; Neidle, S., Warning, M., Eds.; Macmillan: London
1994. (b) Wang, L.; Price, H. L.; Juusola, J.; Kline, M.; Phanstiel, O., IV
J. Med. Chem. 2001, 44, 3682-3691. (c) Lee, Y.-A.; Lee, S.; Cho, T.-S.;
Kim, C.; Han, S. W.; Kin, S. K. J. Phys. Chem. B 2002, 106, 11351-
11355.
* To whom correspondence should be addressed. Phone: 55 51 3320
3629 and 55 51 3308 6321.
† CPBMF.
‡ UFRGS Porto Alegre.
(1) (a) Wang, J. Nucleic Acids Res. 2000, 28, 3011-3016. (b) Niemeyer,
C. M.; Blohm, D. Angew. Chem., Int. Ed. 1999, 38, 2865-2869.
(2) (a) Pu, L. Chem. ReV. 2004, 104, 1687-1716. (b) Valeur, B.
Molecular Fluorescence: Principles and Applications; Wiley-VCH: Wein-
heim, 2002.
(3) Prentø, P. Biotech. Histochem. 2001, 76, 137-161.
(4) Cantor, C. R.; Schimmel, P. R. In Biophysical Chemistry; W. H.
Freeman and Co.: San Francisco, 1980; Part II, pp 392-463.
(6) Viola, G.; Acqua, F. D.; Gabellini, N.; Moro, S.; Vedaldi, D.; Ihmels,
H. ChemBioChem 2002, 3, 550-558.
(7) For a review, see: Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem.
ReV. 1999, 99, 2777-2795.
(8) Cholody, W. M.; Kosakowka-Cholody, T.; Hollingshead, M. G.;
Hairprakasha, H. K.; Michejda, C. J. J. Med. Chem. 2005, 48, 4474-4481.
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1254-1267.
10.1021/ol701708y CCC: $37.00
© 2007 American Chemical Society
Published on Web 09/05/2007

backs especially in electrophoresis purification, due to the
concomitant migration of the cation and the anion along the
gel. Therefore, the DNA bands in the agarose gel some-
times get poorly resolved as in the case of the commercially
available ethidium bromide, which is widely used for DNA
visualization in agarose gel electrophoresis.12 Furthermore,
the design of new small molecular organic fluorophores is
not a simple task.9 Higher selectivity, sensitivity, shorter
assay times, and greater simplicity in performing the assay
are trends that must be taken into account in the design of
new intercalators that may be commercially viable.13
Neutral and highly polar dyes as DNA intercalators are
rare examples,14 but they can be also used for visualization
of biomacromolecules. In this respect, fluorophores such as
quinoxalines,15 benzimidazoles,16 and 2,1,3-benzothiadia-
zoles17 have widespread use in scientific and technological
areas,18 mainly in light technology applications.19 In this
respect, we have recently shown that the photophysical
properties of 2,1,3-benzothiadiazoles (BTD) can be tuned
by proper choice of the nature of the π-extension in positions
4 and 7.20-22
synthesized combining both Sonogashira and Suzuki cou-
pling reactions (see the Supporting Information for details).
The photophysical properties of compounds 1a-c, 2a,b,
and 3a-c were performed in phosphate buffer solutions (100
mM, pH ) 7.0), and the results obtained are summarized in
Table 1.
Table 1. UV-vis and Fluorescence of the BTD Dyes 1a-c,
2a,b, and 3a-c in Phosphate Buffer 100 mM (pH ) 7.0)
Stokes shift
λmabasx (nm) λmemax (nm)
(nm)
Φf
a
dye
log ꢀ (ꢀ)
1a 4.35 (22342)
1b 3.76 (5790)
365
429
438
411
367
444
401
426
471
563
552
535
506
544
547
525
106
134
114
124
139
100
146
99
0.86
0.29
0.37
0.51
0.80
0.40
0.44
0.47
1c
3.81 (6482)
2a 3.22 (1644)
2b 3.33 (2150)
3a 3.97 (9152)
3b 3.78 (6076)
3c
4.01 (10208)
a Quantum yield of fluorescence [quinine sulfate in 1 M H2SO4, f )
0.55, as standard].
These molecules possess the geometry and electronic
properties desirable for DNA duplex intercalators. Here, we
demonstrate that new neutral and highly polar BTD are
outstanding light up probes for human DNA.
In order to check both electronic and geometrical param-
eters on the properties of BTD as light up probes to DNA,
we have changed the groups attached to the 4,7-BTD core
(Chart 1).
In phosphate buffer (100 mM, pH ) 7.0), the lowest
energy absorption bands for compounds 1a-c, 2a,b, and
3a-c (see the Supporting Information) are assigned to π-π*
transitions by virtue of their large molar extinction coef-
ficients (log ꢀ values in the range of 3.22-4.35). The
max
abs
max
em
absorption (λ ) and emission (λ ) maxima lie between
365 and 444 nm and 471-563 nm, respectively. It is worth
noting that all dyes (1a-c, 2a,b, and 3a-c) have large Stokes
shifts in solution, 99-146 nm, allowing unambiguous
detection without reabsorption effects and not interfering with
the background fluorescence of biomolecules. These high
values also indicate a very efficient intramolecular charge
transfer (ICT) in the excited state between the terminal
aromatic group (phenyl ring or a methoxyphenyl group) and
the BTD moiety.
Chart 1. Tested BTD Dyes 1a-c, 2a,b, and 3a-c
The dyes 1a-c and 2a,b were synthesized using a
methodology previously described.20,22 The BTD 3a-c were
Spectrophotometric Titrations. All synthesized dyes
max
abs
showed λ in the near-UV region of the spectrum (∼390
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(14) For example, see: Valis, L.; Enthart, E. M.; Wagenknecht, H-A.
Bioorg. Med. Chem. Lett. 2006, 16, 3184-3187.
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Zhang, X. H. Synth. Met. 1999, 105, 141-144.
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4933.
nm), well separated from the one of the nucleic bases (∼260
nm). Negative results were obtained during titration using
the systems 2a,b since it is necessary to use a high concen-
tration of the intercalating agent and high concentrations of
DNA. However, all compounds having a CtC π spacer
1a-c and especially 3a-c, were successfully tested as sensi-
tive probes for selective DNA detection (Figure 1(A) and
Figures S4 and S6, Supporting Information). This fact indi-
cated the necessity of the triple bound spacer CtC in order
to facilitate the intercalation binding between the dyes and
DNA duplex. All compounds (1a-c, 2a,b, and 3a-c) were
also tested against human and Mycobacterium tuberculosis
purine nucleoside phosphorylase (PNP) enzymes with nega-
(21) Neto, B. A. D.; Lopes, A. S.; Wust, M.; Costa, V. E. U.; Ebeling,
G.; Dupont, J. Tetrahedron Lett. 2005, 46, 6843-6846.
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4002
Org. Lett., Vol. 9, No. 20, 2007

wavelength absorption maxima was observed for compound
3a, 2-5 nm for 3b, and impressive 5-19 nm for BTD 3c.
To the best of our knowledge, these series (3a-c) are among
the most sensitive light up probes for DNA detection using
spectrophotometric titrations reported to date.
Spectrofluorimetric Titrations. Spectrofluorimetric titra-
tions of DNA to the BTD derivatives 1a-c, 2a,b, and
3a-c were performed in an aqueous buffer solution at a
ligand concentration of 10 µM. Under these conditions, no
precipitation was observed. No significant results were
obtained using the BTD systems 2a,b, which indicates that
even in a more sensitive technical analysis (fluorescence),
the presence of a CtC π spacer is fundamental to allow
the binding of the dye to the DNA duplex. All compounds
(1a-c, 2a,b, and 3a-c) were equally tested against human
and M. tuberculosis purine nucleoside phosphorylase (PNP)
enzymes with negative results indicating once more their
sensitivity and selectivity to DNA.
Compounds 1a-c showed interesting results (see Figure
S6 in the Supporting Information). In particular, 1b presents
a very different behavior than all other BTD. This compound
decomposes upon irradiation at 429 nm. Compounds 1a and
1c, however, apparently could be useful as light up probes
to DNA detection using fluorimetric titration. In both cases,
the dyes used in very low concentration (10 µM) could detect
even 1 ppm of DNA in phosphate buffer solutions. Never-
theless, after a few weeks in the stock solution, we observe
degradation of compound 1c. Compound 1a presented its
long wavelength emission maximum below 500 nm (473 nm
for 1a, see Table 1) and therefore not suitable for the
detection of fluorescence without distortion by autofluores-
cence of the cell matrix.23 However, dyes 3a-c present the
best results for fluorescence detection and quantification
(Figure 1 (B) and Figures S4 and S6). The insertion of one
4-MeOPh group on the molecular architecture of compounds
3a-c increases the thermal, electrochemical, and excited-
state stability of the small fluorescent organic systems.
Figure 1. Spectrophotometric (A) and Spectrofluorimetric (B)
titrations of DNA to compound 3a (10 µM in a phosphate buffer
(100 mM) in all measurements) (ppm ) ng/mL).
tive results, indicating their sensitivity and selectivity toward
DNA.
The addition of DNA (10-100 ppm) to the buffered
solutions of the BTD 1a (50 µM) causes a significant
decrease of the absorbance (hypochromic effect) and a blue
shift (3-5 nm) of the long wavelength absorption maxima.
The exponential decay of the relative absorbance intensity
furnishes a quantitative detection of the DNA using BTD
1a. Note that using spectrophotometric titration, this com-
pound is the only one that acts as a “light off” probe, and
this clearly indicates the importance of a donor group
(PhOMe) attached to the BTD core.
Compound 1b (50 µM) is not a good candidate due to the
pronounced rotational bands that result in a poorly resolved
spectra. Moreover, it indicates that the interaction occurs,
increasing the absorbance intensity exponentially upon
binding with DNA, and the correlation with the biomacro-
molecules is almost perfect. Among compounds 1a-c, BTD
1c showed the best results using spectrophotometric titration.
Upon addition of DNA (5-75 ppm) to the buffered solutions
of the BTD 1c (50 µM), a significant increase of the
absorbance (hyperchromic effect) and a red shift (5-12 nm)
of the long wavelength absorption maxima was observed.
The dyes 3a-c gave the best results among all compounds
(Figure 1(A) and Figures S4 and S6). The molecular arch-
itecture designed for these sensitive dyes indicates the
efficiency of our strategy of combining in the organic systems
a donating group (4-MeOPh) directly attached to the BTD
nucleus on one side and the presence of CtC π spacer on
the other.
In all cases, the dyes used in very low concentration (10
µM) could detect even 1 ppm of DNA in phosphate buffer
solutions. BTD 3a showed a significant increase of the
fluorescence intensity (hyperchromic effect) and a red shift
(2-5 nm) of the long-wavelength emission maximum. The
exponential increase of the relative fluorescence intensity
enabled us to carry out a quantitative detection of the
DNA using BTD 3a in an almost perfect match as seen in
Figure 1.
Compounds 3b and 3c presented similar results with a
significant increase in the fluorescence intensity and a slight
red shift (1-4 nm and 2-3 nm, respectively) of the long
wavelength emission maxima. Both fluorescent systems also
presented an exponential increase of fluorescence upon
increasing the DNA concentration. It is interesting for a
comparison that commonly used florescent probes normally
possess a detection limit that is equal to or above 10 ppm24
(see Table T1 in the Supporting Information) such as the
widely used ethidium bromide.24a In fact, BTD 3a-c
Compounds 3a-c possess high sensitivity to DNA detec-
tion, i.e., down to 1 ppm of DNA (the hyperchromic effect
observed were very pronounced, especially observed for
compound 3c). A red shift between 2 and 7 nm of the long
(23) Patonay, G.; Antoine, M. D. Anal. Chem. 1991, 63, 321A-327A.
Org. Lett., Vol. 9, No. 20, 2007
4003

fluorescent light up probes, especially, are among the most
sensitive probes ever described.
Upon binding with DNA, compounds 3a-c maintain their
high Stokes shift, with a red shift after the association with
DNA, indicating a more efficient ICT process. If the
association proceeded through the electron-donating group,
the ICT process would not be expected to be so efficient.
All results indicate that the intercalation occurs at the CtC
side of the BTD, and as a consequence, the PhOMe portion
would be free to perform the ICT process with the BTD core,
as rationalized in Figure 2. Preliminary stopped-flow kinetic
Figure 3. UV irradiation at 360 nm: (A) dye 3a (1 µL of 1 mM
solution) and (B) ethidium bromide (1 µL of 1 mM solution). Gel
channels, left to right: control, 1 Kb plus; pCINeo, 200 ng; pCINeo,
100 ng; pCINeo, 50 ng; pCINeo, 20 ng; pCINeo, 10 ng; pCINeo,
5 ng.
In summary, BTDs containing a PhOMe donating group
on one side associated with an Ar-CtC group is a suitable
molecular architecture for the spectroscopic selective detec-
tion and quantification of DNA. The new dyes are among
the most sensitive probes for the DNA detection in solution
by spectrophotometric and spectrofluorimetric titrations. A
full intercalating model study with completely kinetic and
binding model with stopped-flow, surface plasmon reso-
nance, real time PCR, electrochemical, and theoretical
approach will be published in due course.
Figure 2. Proposed architecture upon DNA binding.
experiments indicate high binding constants (immediately
binding to DNA).
Acknowledgment. We thank CAPES and CNPq for
A comparative and qualitative experiment of resolution
and sensitivity to DNA was performed with dye 3a,
comparing with the commercially available ethidium bromide
(Figure 3). The commercially available DNA plasmid
(pCINeo) at different concentrations (200, 100, 50, 20, 10,
5 ng) was submitted in two different agarose gels (1%) to
electrophoresis purification using commercial 1 kb plus as
a control. It is clear from Figure 3 that the agarose gel A
(used with compound 3a) reveals DNA in the six different
concentrations, while agarose gel B (used with ethidium
bromide) reveals only the most concentrated DNA channel
(200 ng). It is also worth noting that there is no migration
of compound 3a, as commonly occurs using charged dyes
such as ethidium bromide. We can also note that using
compound 3a, we were able to reveal the bands with small
mass (see the control band in Figure 3), while it is not
possible with ethidium bromide (see Supporting Information
for 3b).
financial support.
Supporting Information Available: Synthetic and titra-
tion experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL701708Y
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