Cyanines as new fluorescent probes for DNA detection and two-photon excited bioimaging

Article abstract of DOI:10.1021/ol100474b

A series of cyanine fluorophores based on fused aromatics as an electron donor for DNA sensing and two-photon bioimaging were synthesized, among which the carbazole-based biscyanine exhibits high sensitivity and efficiency as a fluorescent light-up probe for dsDNA, which shows selective binding toward the AT-rich regions. The synergetic effect of the bischromophoric skeleton gives a several-fold enhancement in a two-photon absorption cross-section as well as a 25- to 100-fold enhancement in two-photon excited fluorescence upon dsDNA binding.

Full text of DOI:10.1021/ol100474b

ORGANIC
LETTERS
2010
Vol. 12, No. 10
2194-2197
Cyanines as New Fluorescent Probes
for DNA Detection and Two-Photon
Excited Bioimaging
Xin Jiang Feng,†,‡ Po Lam Wu,†,§ Fre´de´ric Bolze,⊥ Heidi W. C. Leung,‡
King Fai Li,†,§ Nai Ki Mak,| Daniel W. J. Kwong,‡ Jean-Franc¸ois Nicoud,*,⊥
Kok Wai Cheah,*,†,§ and Man Shing Wong*,†,‡
Centre for AdVanced Luminescence Materials, Department of Chemistry,
Department of Physics, and Department of Biology, Hong Kong Baptist UniVersity,
Kowloon Tong, Hong Kong SAR, China, and Laboratoire de Biophotonique et
Pharmacologie, Faculte´ de Pharmacie (UMR 7213), UniVersite´ de Strasbourg,
74 route du Rhin, F-67401 Illkirch, France
mswong@hkbu.edu.hk; kwcheah@hkbu.edu.hk; jfnicoud@unistra.fr
Received February 26, 2010
ABSTRACT
A series of cyanine fluorophores based on fused aromatics as an electron donor for DNA sensing and two-photon bioimaging were synthesized,
among which the carbazole-based biscyanine exhibits high sensitivity and efficiency as a fluorescent light-up probe for dsDNA, which shows
selective binding toward the AT-rich regions. The synergetic effect of the bischromophoric skeleton gives a several-fold enhancement in a
two-photon absorption cross-section as well as a 25- to 100-fold enhancement in two-photon excited fluorescence upon dsDNA binding.
There has been considerable interest in the development of
facile and reliable methods to detect nucleic acids owing to their
potential applications in bioanalytical, clinical, and forensic
analysis.1 Fluorophores that show an increase in fluorescence
intensity upon binding with DNA have been used to stain and
quantify nucleic acids in gel electrophoresis as well as in staining
cell nuclei for microscopic bioimaging.2 In addition, fluorescent
dyes that possess high two-photon absorption (TPA) properties
may be useful in two-photon excited fluorescence (TPEF)
microscopic imaging.3 As the two-photon absorption process
is quadratically intensity-dependent, the excitation is confined
to a small volume in the focal plane, which can provide very
high spatial resolution with greatly reduced photobleaching and
photodamage to the sample speciment during imaging. Fur-
thermore, the use of an infrared laser source improves light
† Centre for Advanced Luminescence Materials, Hong Kong Baptist
University.
‡ Department of Chemistry, Hong Kong Baptist University.
§ Department of Physics, Hong Kong Baptist University.
⊥ Faculte´ de Pharmacie (UMR 7213), Universite´ de Strasbourg.
| Department of Biology, Hong Kong Baptist University.
(1) (a) Wang, J. Nucleic Acid Res. 2008, 28, 3011. (b) Umek, R. M.;
Lin, S. W.; Vielmetter, J.; Terbrueggen, R. H.; Irvine, B.; Yu, C. J.; Kayyem,
J. F.; Yowanto, G. F.; Farkas, D. H.; Chen, Y. P. J. Mol. Diagn. 2001, 3,
74. (c) Dubertret, B.; Calame, M.; Libchaber, A. Nat. Biotechnol. 2001,
19, 365. (d) Pu, K.-Y.; Liu, B. AdV. Funct. Mater. 2009, 19, 1.
(2) (a) Jin, L.-T.; Choi, J.-K. Electrophoresis 2004, 25, 2429. (b) Herr,
A. E.; Singh, A. K. Anal. Chem. 2004, 76, 4727.
(3) Zipfel, W. R.; Williams, R. M.; Webb, W. W. Nat. Biotechnol. 2003,
21, 1369.
10.1021/ol100474b  2010 American Chemical Society
Published on Web 04/20/2010

penetration depth in tissues, thus allowing deeper tissue imaging.
However, most of the currently used one-photon excited
biological fluorophores show only low TPA cross sections (σ)
and/or TPEF brightness (ΦFLσ).4 Moreover, most of the highly
active TPA molecules known are not biocompatible as they
are often water-insoluble or form nonfluorescent aggregates in
aqueous media. Consequently, highly sensitive two-photon
excited fluorophores for DNA are rather rare.5
using HIO4 and I2 in acetic acid gave diiodo-product, 1a in 66%
yield. Palladium-catalyzed Heck-coupling of the diiodide 1a
and 4-vinylpyridine using Pd(OAc)2/2P(o-tol)3 as a catalyst
afforded bis-product 2a in 45% yield. Subsequent methylation
carried out in DMF in the presence of CH3I at 90 °C gave
dibenzofuran-based biscyanine, V-furan, in 71% yield. Fol-
lowing the same reaction sequence, di-iodination of diben-
zothiophene (31%), Heck-coupling of the diiodide compound with
4-vinylpyridine (70%) and methylation (78%), and dibenzothiophene-
based biscyanine, V-thiophene was also synthesized.
To enhance the water solubility and promote binding
propensity with the negatively charged DNA6 as well as
induce the large two-photon absorption properties,7 novel
bis-cationic donor-acceptor fluorophores based on electron-
donating fused aromatics, including dibenzothiophene, diben-
zofuran, and carbazole, were designed and investigated for
DNA detection and two-photon excited bioimaging. In this
contribution, we have demonstrated that multifunctional
carbazole-based biscyanine is a highly sensitive and efficient
fluorescent light-up probe for double-stranded DNA (dsDNA)
with a binding selectivity toward the AT-rich region. The
synergetic effect of the bischromophore leads to a large
enhancement in the TPA cross-section of biscyanine as
compared to that of monocyanine. In addition to the several-
fold increase in TPA cross-section, the carbazole-based cyanines
exhibit 25-100-fold enhancement in TPEF upon dsDNA
association, highlighting their potential as highly sensitive TPEF
probes for DNA detection and fluorophores for bioimaging.
Initial TPEF imaging study of the carbazole-based cyanine
fluorophores in HeLa cells showed bright images with high
contrast at low laser powers and specific subcellular localizations
with no discernible photobleaching effects.
On the other hand, the Knoevenagel reaction of carbazolyl-
3,6-dialdehyde and N-methyl-4-methyl-pyridium iodide was
used to synthesize carbazole-based biscyanine V-carbazole and
monocyanine S-carbazole. Alkylation of carbazole with eth-
ylene glycol tosylate in the presence of NaOH gave alkylated
carbazole 3a in an excellent yield. Dibromination of 3a in the
presence of NBS gave alkylated 3,6-dibromocarbazole, 4a, in
91% yield. Double formylation of 4a via lithiation bromide
exchange at low temperature followed by the subsequent
reaction with N-formylmorpholine afforded carbazolyl-3,6-
dialdheyde 5a in 48% yield. Knoevenagel reaction of the
dialdehyde 5a and N-methyl-4-methylpyridium iodide in the
presence of piperidine in ethanol afforded carbazole-based bis-
cyanine, V-carbazole in a good yield (72%). S-carbazole was
also prepared in a similar fashion. All the newly synthesized
biscyanines were fully characterized with 1H NMR, 13C NMR,
MS, and elemental analysis and found to be in good agreement
with the proposed structures. Thermogravimetric analysis
showed that these biscyanines possess high decomposition
temperatures ranging from 314 to 343 °C.
Palladium-catalyzed Heck coupling of 2,7-diiododibenzofuran
or 2,7-diiododibenzothiophene and 4-vinyl-pyridine using Pd-
(OAc)2/2P(o-tol)3 as a catalyst was used as a key step to
synthesize dibenzofuran- and dibenzothiophene-based biscya-
nines, respectively (Scheme 1). Di-iodination of dibenzofuran
All the biscyanines are highly soluble in aqueous solution
with no aggregation. The aqueous solutions of V-carbazole
obeyed Beers’s law up to a concentration of 1 × 10-4 M. As
shown in Figure 1, all the biscyanines show very similar
Scheme 1. Synthesis of Fused Aromatic-Based Cyanines
Figure 1. (a) Absorption spectra of biscyanines without and with
5 equiv of calf thymus DNA (ct-DNA) measured in pH 7 phosphate
buffer solution. (b) Fluorescence spectra of V-carbazole upon
addition of various concentrations of ct-DNA.
absorption characteristics, featuring a strong, broad, and struc-
abs
tureless low-energy absorption band (λ ) at around 369-436
max
(4) (a) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B 1996, 13, 481. (b)
Hayek, A.; Bolze, F.; Nicoud, J.-F.; Baldeck, P. L.; Me´ly, Y. Photochem.
Photobiol. Sci. 2006, 5, 102. (c) Hayek, A.; Ercelen, S.; Zhang, X.; Bolze,
F.; Nicoud, J.-F.; Schaub, E.; Baldeck, P. L.; Me´ly, Y. Bioconjugate Chem.
2007, 18, 844.
Org. Lett., Vol. 12, No. 10, 2010
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Table 1. Summary of Photophysical Measurements of S-Carbazole, V-Carbazole, V-Thiophene, and V-Furan Measured in Buffer Solution
abs
abs
λ
/nm
λ
/nm
max
max
b c
,
e
a b
,
(εmax 104/M-1cm-1
)
λ
a/nm
ΦFL
τd/ns
(εmax 104/M-1 cm-1
)
λ
a,f/nm
em
max
ΦFL
τc,f/ns
em
max
without ct-DNA
with addition of ct-DNA
S-carbazole
V-carbazole
V-thiophene
V-furan
416 (4.03)
436 (5.81)
375 (3.40)
369 (2.69)
573
573
535
467
0.02
1.95
0.78
0.48
418 (3.79)
451 (4.98)
391 (3.13)
374 (2.36)
563
548
511
449
0.07
0.13
0.093
<10-3
2.37
2.34
2.18
0.0029
0.031
<10-3
f
g
-
-
a Excited at the absorption maximum b Using coumarin 6 (Φ420 ) 0.78) as a standard for S-carbazole and V-carbazole and quinine sulfate monohydrate
(ΦO350 ) 0.58) as a standard for V-thiophene and V-furan. c Average of two independent measurements. d Fluorescence lifetime e 5 equiv of calf thymus
DNA (ct-DNA) added. f 100 equiv of ct-DNA added. g Too low to be determined reliably.
nm in phosphate buffer solution corresponding to the π-π*
DNA, increasing from 0.003 without ct-DNA to 0.13 at
saturation, highlighting its potential as a fluorescence light-
up probe for DNA detection. To ascertain the application of
V-carbazole for DNA quantitation, fluorescence titration was
performed using the pcDNATM6.2-GW/EmGFP-miR-neg
plasmid DNA (Supporting Information). Linear variation of
fluorescence intensity was observed from 60 ng/L of DNA
down to a few ng/L at a dye concentration of 10-7 M with
a slope of 1189. Thus, this dye offers a straightforward way
to quantify DNA concentration by fluorescence technique.
The detailed binding characteristics of the biscyanine,
V-carbazole, with DNA were investigated, together with its
monocyanine counterpart, S-carbazole, for comparison,
using various defined-sequence oligonucleotides. S-carba-
zole shows similar spectral responses upon DNA binding
which included a red absorption peak shift, a blue emission
peak shift, a decrease in fluorescence lifetime, and an increase
in fluorescence quantum yield, albeit to a lesser extent
supporting the merit of using the bis-cationic approach for
DNA detection. In these fluorescence titrations, both cyanines
exhibited higher sensitivity toward dsDNA than the corre-
sponding single-stranded DNA (ssDNA), particularly for
V-carbazole. They also show much larger fluorescence
enhancement toward AT-rich dsDNA, suggesting selective
binding toward the AT-rich regions. Weaker fluorescence
enhancement on GC-rich sequences might be caused by
fluorescence quenching properties of guanine. The binding
associations (Kb) of V-carbazole with DNA d[AT]5:d[TA]5
and DNA d[A]10:d[T]10 as estimated by the nonlinear curve
fitting analysis are 8.3 × 105 M-1 and 20.5 × 105 M-1,
respectively, which are much larger than that obtained with
DNA d[C]15:d[G]15 (Kb ) 1.5 × 105 M-1). These results
are consistent with the fact that the environments of the AT-
rich sites in dsDNA are relatively electronegative, which
favors binding interaction with the positively charged dyes.
On the other hand, the exocyclic NH2 substituent of guanine
in the GC base pair regions has been reported to hinder
effective binding of a dye.8 In contrast, the binding associa-
tions of S-carbazole with DNA d[AT]5:d[TA]5 and DNA
d[A]10:d[T]10 are 1.1 × 104 M-1 and 4.7 × 104 M-1,
respectively, which are substantially smaller than those of
V-carbazole. These findings are also in good agreement with
transition due to their large molar absorptivities (Table 1).
abs
Upon excitation at their λ , these biscyanines showed a
max
very weak emission at ∼467-573 nm (fluorescence quantum
yield, ΦFL ) 0.001-0.031) in sharp contrast to the moderate
emission obtained in an organic solvent (ΦFL ) 0.001-0.16
in DMSO). There are large Stokes shifts between the
absorption and emission spectra (∆98-160 nm) among these
biscyanines, indicating that their first excited states, arising
from the intramolecular charge transfer, are highly stabilized
by the aqueous medium.
Upon addition of calf thymus DNA (ct-DNA) to the
biscyanine buffered solutions, significant red shifts of the
abs
absorption spectra (∆λ ) 5-16 nm) with a concomitant
max
decrease in the absorptivities were observed (Figure 1a),
indicating an association of these V-shaped cationic dyes
with DNA. Remarkably, despite very weak emission in a
buffer medium, there is a progressive and dramatic increase
in fluorescence intensity together with blue shift of emission
spectra upon titration of these biscyanines with ct-DNA.
(Figure 1b). The fluorescence enhancement is attributed to
the large reduction in the nonradiative decay of the photo-
excited biscyanines due to the restricted rotation of the central
C-C bond upon DNA binding. This explanation is consistent
with the observed increase in the fluorescence lifetimes of
these biscyanines upon addition of ct-DNA (e.g.: V-carba-
zole, τ ) 0.78 ns, increased to τ ) 2.34 ns in the presence
of ct-DNA) and in the ΦFL in a viscous solvent (e.g.: ΦFL
of V-carbazole in 100 g/L of polyethyleneglucol-5000 )
0.016).5b It is important to note that V-carbazole exhibits
much stronger (77-fold) fluorescence enhancement at satura-
tion with ct-DNA than V-thiophene (2-fold) and V-furan
(5-fold). There is also ∼40 times increase in the fluorescence
quantum yield (ΦFL) for V-carbazole at saturation with ct-
(5) (a) Abbotto, A.; Baldini, G.; Beverina, L.; Chirico, G.; Collini, M.;
D’Alfonso, L.; Diaspro, A.; Magrassi, R.; Nardo, L.; Pagani, G. A. Biophys.
Chem. 2005, 114, 35. (b) Allian, C.; Schmidt, F.; Lartia, R.; Bordeau, G.;
Fiorini-Debuisschert, C.; Charra, F.; Tauc, P.; Teulade-Fichou, M.-P.
ChemBioChem 2007, 8, 424.
(6) Ihmels, H.; Otto, D. Top. Curr. Chem. 2005, 258, 161.
(7) (a) Rumi, M.; Ehrlich, J. E.; Heikal, A. A.; Perry, J. W.; Barlow,
S.; Hu, Z.; McCord-Maughon, D.; Parker, T. C.; Ro˜ckel, H.; Thayumanavan,
S.; Marder, S. R.; Beljonne, D.; Bre´das, J. L. J. Am. Chem. Soc. 2000, 122,
9500. (b) Mongin, O.; Porre`s, L.; Charlot, M.; Katan, C.; Blanchard-Desce,
M. Chem.sEur. J. 2007, 13, 1481. (c) Lo, P. K.; Li, K. F.; Wong, M. S.;
Cheah, K. W. J. Org. Chem. 2007, 72, 6672.
(8) Cameron, L.; Thompson, A. Biochemistry 2000, 39, 5004.
Org. Lett., Vol. 12, No. 10, 2010
2196

the plasmid DNA unwinding assay,9 which showed that
S-carbazole is a DNA-intercalator while V-carbazole
exhibits no DNA-intercalating property and thus prefers to
bind within the minor groove (Supporting Information).
Both carbazole-based cyanines exhibit interesting two-
photon-induced photoluminescence at around 575-578 nm
upon excitation at 800 nm femtosecond (fs) laser pulses in
buffered solution, which are slightly red-shifted as compared
to the one-photon emission spectra because of reabsorption
effect, suggesting that the same emissive states are involved
in the two excitation processes. The TPA cross sections (σ)
and the two-photon excited spectra of the carbazole-based
cyanines were determined by the two-photon induced
fluorescence method without and with ct-DNA in phosphate
buffer solution.10 Figure 2 shows the two-photon excitation
Figure 3. (a) TPEF image of V-carbazole in HeLa cells excited at
800 nm fs laser pulses using 10 mW laser power. (b) FLIM image
of the same cell measured by the TCSPC method with lifetime
distribution.
Fluorescence lifetime imaging microscopic (FLIM) experi-
ments showed that this dye exhibits good photobleaching
resistance and specific localization in the cell cytoplasm as
measured by the time-correlated single photon counting
(TCSPC) method (Figure 3b). Apparently, the nuclear
penetration of V-carbazole was not very efficient, resulting
in a brighter signal seen at the cytoplasm than in the nucleus.
Nevertheless, two distinct subcellular environments, indicated
by the fluorescence lifetimes, were observed for this dye: in
the perinuclear region, τ ) 2.0 ns, and in the heterogeneously
stained cytoplasm, where τ ) 1.2 ns. Interestingly, the TPEF
image of S-carbazole in Hela cells was obtained with a lower
laser power (5 mW) than that for V-carbazole producing
the same quality image, indicating the higher sensitivity of
S-carbazole for TPEF bioimaging (Supporting Information).
Figure 2. Two-photon excitation spectra of (a) V-carbazole and
(b) S-carbazole without and with 200 equiv of ct-DNA in 10-5
M
buffered solution.
In summary, we have synthesized a series of cyanine
fluorophores based on fused aromatics as an electron donor for
DNA quantitation and two-photon bioimaging, among which
the carbazole-based biscyanine exhibits high sensitivity and
efficiency as a fluorescent light-up probe for dsDNA. The bis-
chromophoric skeleton gives a several-fold enhancement in the
TPA cross-section and 25- to 100-fold enhancement in TPEF
brightness upon dsDNA binding, making these carbazole-based
cyanines highly sensitive TPEF probes for DNA detection in
fluorescence bioassays. The TPEF images of the carbazole-
based cyanine fluorophores in HeLa cells exhibit good bright-
ness and high contrast at low laser powers. These cyanine dyes
also show specific subcellular localization without detectable
photobleaching effects. Further studies are underway to deter-
mine the specific subcellular localizations of these dyes.
spectra of V-carbazole and S-carbazole with σmax reaching
1241 GM at 830 nm for V-carbazole and 431 GM at 850
nm for S-carbazole, respectively, demonstrating the syner-
getic effect of the bis-chromophoric skeleton in enhancing
the nonlinear optical properties. There are also dramatic
enhancements (25-100-fold) of TPEF brightness of these
cyanines upon addition of ct-DNA up to 284 GM and 218
GM for V-carbazole and S-carbazole, respectively, which
are among the highest TPEF brightness values reported with
DNA bioaffinity.5b In the presence of 200 equiv of ct DNA,
the fluorescence quantum yields were enhanced, and the TPA
cross sections increased greatly up to 2170 GM at 810 nm
and 3127 GM at 880 nm for V-carbazole and S-carbazole,
respectively. The present results on fluorescence light-up
upon DNA binding are of great interest in fluorescence
bioassays, which can take great advantages of the two-photon
excitation and enable bioaffinity experiments without separa-
tions in a complex medium with subpicomolar sensitivity.11
An initial TPEF microscopic study of the carbazole-based
cyanine fluorophores in Hela cells was carried out using 800
nm fs laser pulses. Figure 3a depicts the TPEF image of
HeLa cells incubated with 10-6 M V-carbazole (10 mW
laser power at sample) showing good contrast and brightness.
Acknowledgment. This work was financially supported by
Hong Kong Research Grant Council, GRF, HKBU 202408,
Hong Kong Baptist University, the Faculty Research Grant,
FRG/08-09/II-16, and the PROCORE-France/Hong Kong Joint
Research Scheme sponsored by the Research Grants Council
of Hong Kong and the Consulate General of France in Hong
Kong, F-HK04/07T. L. Richert and Y. Me´ly at UdS are
acknowledged for two-photon microscopy experiments.
Supporting Information Available: Detailed synthetic
procedures, results of physical characterization, and spectral
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
(9) Munson, B. R.; Fiel, R. L. Nucleic Acids Res. 1992, 20, 1315.
(10) Wu, P. L.; Xia, P. F.; Li, Z. H.; Feng, X. J.; Tam, H. L.; Li, K. F.;
Jiao, Y.; Wong, M. S.; Cheah, K. W. Chem. Commun. 2009, 5421.
(11) (a) Ha¨nninen, P.; Soukka, J.; Soini, J. T. Ann. N.Y. Acad. Sci. 2008,
1130, 320. (b) Ha¨nninen, P.; Soini, A.; Meltola, N.; Soini, J.; Sukka, J.;
Soini, E. Nat. Biotechnol. 2000, 18, 548.
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