Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn-On DNA Probes

Article abstract of DOI:10.1002/asia.201601430

Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine

Full text of DOI:10.1002/asia.201601430

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Key Structural Elements of Unsymmetrical Cyanine Dyes for
Highly Sensitive Fluorescence Turn-on DNA Probe
Authors: Kakishi Uno, Taeko Sasaki, Nagisa Sugimoto, Hideto Ito,
Taishi Nishihara, Shinya Hagihara, Tetsuya Higashiyama,
Narie Sasaki, Yoshikatsu Sato, and Kenichiro Itami
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Asian J. 10.1002/asia.201601430
Link to VoR: http://dx.doi.org/10.1002/asia.201601430
A Journal of
A sister journal of Angewandte Chemie
and Chemistry – A European Journal

10.1002/asia.201601430
Chemistry - An Asian Journal
FULL PAPER
Key Structural Elements of Unsymmetrical Cyanine Dyes for
Highly Sensitive Fluorescence Turn-on DNA Probe
Kakishi Uno,[1] Taeko Sasaki,[1] Nagisa Sugimoto,[2] Hideto Ito,[1] Taishi Nishihara,[1,3] Shinya
Hagihara,[1,2] Tetsuya Higashiyama,[1,2,4] Narie Sasaki,[1] Yoshikatsu Sato,[2] and Kenichiro Itami*[1,2,3]
Abstract: Unsymmetrical cyanine dyes such as thiazole orange are
useful for detecting nucleic acids with fluorescence because they
dramatically enhance the fluorescence upon binding to nucleic acids.
In this paper, we synthesized a series of unsymmetrical cyanine
dyes and evaluated their fluorescence properties. A systematic
structure-property relationship study has uncovered that the
dialkylamino group at the 2-position of quinoline in a series of
unsymmetrical cyanine dyes plays a critical role in the fluorescence
enhancement. Four newly designed unsymmetrical cyanine dyes
showed negligible intrinsic fluorescence in a free-state and strong
fluorescence upon binding to double-stranded DNA (dsDNA) with
quantum yield 0.53−0.90, which is 2-3 times higher than the
previous unsymmetrical cyanine dyes. Detailed analysis of
fluorescence lifetime revealed that dialkylamino group at the 2-
position of quinoline suppressed non-radiative decay in favor of
increasing fluorescence quantum yield. Moreover, these newly
developed dyes were able to specifically stain the nucleus in fixed
HeLa cells with a confocal laser-scanning microscope.
use in nucleic acid imaging such as strong absorptivity, high
brightness, and high water solubility. More importantly, vast
majority of those probes show very low fluorescence in aqueous
media but turn to be fluorescent state with strong brightness
upon binding to nucleic acids. This particular character,
according to many reports, is caused by the fixation of the
cyanine conjugate upon binding to nucleic acids, resulting in the
suppression of non-radiative path from the excited state.[10]
Along with the advances in optical microscopy and biological
technology, the development of unsymmetrical cyanine dyes
with superior properties has been an active research area. The
absorbance of unsymmetrical cyanine dyes has been modified
to fit conventional laser sources. By simply changing the number
of methine unit[11] or the substituents around terminal nitrogen
atoms[12], their absorption regions can be tunable between
ultraviolet and near-infrared region. The fluorescence sensitivity
can be improved by the homodimerization of unsymmetrical
cyanine dyes, which are known as TOTO and YOYO series dyes
(Figure 1).[13] Although these dyes have been used in many
research fields,[14] they still suffer from low fluorescence
dsDNA
quantum yield upon binding to double-stranded DNA (ΦF
)
dsDNA
and poor cell permeability.[15] For example, the ΦF
of
Introduction
TOPRO-3 and TOTO-3 which have extended methine unit and
hence long wavelength absorption are reported to be 0.11 and
0.06 respectively.[16]
Detection of nucleic acids by fluorescence probe has been
common and essential technology in molecular and cellular
biology. It has been used in employing gel electrophoresis,[1]
quantitative PCR,[2] flow cytometry,[3] in vivo nucleic acid
staining,[4] and various other applications.[5] To date,
fluorescence turn-on DNA probes such as DAPI,[6] Hoechst,[7]
and organometallic DNA intercalator[8] have been developed.
The most pervasively used DNA probes are unsymmetrical
cyanine dyes.[9] These dyes consist of two different nitrogen-
containing heterocycles which are bridged by more than one
methine unit. They fulfill some basic requirements for practical
N+
N+
N+
N
n
N+
X
n
N+
X
N+
N
n
X
N
TOPRO and YOPRO dyes
n = 0 or 1, X = O or S
TOTO and YOYO dyes
n = 0 or 1, X = O or S
(
Φ
dsDNA = 0.11–0.44)
(Φ
dsDNA = 0.06–0.52)
F
F
[1]
[2]
K. Uno, T. Sasaki, Dr. H. Ito, Dr. T. Nishihara, Dr. S. Hagihara, Prof.
Dr. T. Higashiyama, Dr. N. Sasaki, Prof. Dr. K. Itami
Graduate School of Science, Nagoya University, Nagoya 464-8602,
Japan.
E-mail: itami@chem.nagoya-u.ac.jp (KI)
N. Sugimoto, Dr. S. Hagihara, Prof. Dr. T. Higashiyama, Dr. Y. Sato,
Prof. Dr. K. Itami
Z
N+
N
N+
N
S
Y
n
X
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya
University, Nagoya 464-8601, Japan.
N
N
[3]
[4]
Dr. T. Nishihara, Prof. Dr. K. Itami
PicoGreen (PG): Y = Z = NMe2
dsDNA = 0.53)
JST-ERATO, Itami Molecular Nanocarbon Project, Nagoya
University, Chikusa, Nagoya 464-8602, Japan.
Prof. Dr. T. Higashiyama
JST-ERATO, Higashiyama Live-Holonics Project, Nagoya
University, Chikusa, Nagoya 464-8602, Japan.
This work
n = 0 or 1, X = O or S
dsDNA = 0.53–0.90)
(
Φ
F
SYBR Green I (SG): Y = H, Z = NMe2
dsDNA = 0.80)
(
Φ
F
(
Φ
F
Figure 1. Unsymmetrical cyanine dyes showing turn-on fluorescence and their
fluorescence quantum yield in the presence of dsDNA (ΦFdsDNA).
Supporting information for this article is given via a link at the end of
the document.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/asia.201601430
Chemistry - An Asian Journal
FULL PAPER
In contrast, SYBR Green I (SG) and PicoGreen (PG) show
excellently high ΦF
dialkylamino group at the 2-position of quinoline core is essential
for the highly fluorescent property of SG.
dsDNA
(>0.5)[17] thereby being widely used for
simultaneous labeling of organelle DNA as well as nuclear
DNA.[18] The core fluorophore of these dyes are essentially
thiazole orange (TO)[19] whose ΦFdsDNA is reported to be quite low
(0.22).[20] We conceived that uncovering the molecular basis of
high fluorescence of SG and PG would lead to the rational
design of new fluorescence turn-on DNA probes. In this paper,
we synthesized a series of TO derivatives and studied how SG
and PG express high ΦFdsDNA. In addition, we applied the
Table 1. Photophysical data of TO, TOPh, TONEt2, TOPhNEt2 and SG.
dsDNA [c]
Compound
TO
λabsdsDNA /nm [a]
λemdsDNA /nm [b]
ΦF
509
515
488
491
501
527
530
517
520
522
0.22
0.22
0.79
0.76
0.74
TOPh
essence of SG and PG to the development of new red
dsDNA
TONEt2
TOPhNEt2
SG
unsymmetrical cyanine dyes with superior ΦF
.
Results and Discussion
Tris-EDTA buffer solution (pH 8.0) was used for all optical measurements.
[a] Absorption maximum wavelength of dye-dsDNA complexes. [b]
Uncorrected maximum fluorescence wavelength of dye-dsDNA complexes.
[c] Absolute fluorescence quantum yield of dye-dsDNA complexes.
Initial structural consideration on the relationship
between SG backbone and fluorescence property. PG and
SG have three characteristic substituents on TO: (1) phenyl
group directly bound on a nitrogen atom of quinoline, (2)
dialkylamino group at the 2-position of quinoline, and (3) one or
two dimethylamino groups dangled to the termini of the
dialkylamino chain connected to quinoline. To gain insights into
the effects of these substituents, first of all, we synthesized three
TO derivatives TONEt2, TOPh, and TOPhNEt2, which share the
partial structure of PG and SG (Figure 2). The synthetic
procedure of TONEt2, TOPh and TOPhNEt2 are described in
Scheme 1 and Supporting Information (SI).
Synthesis of dialkylamino-substituted oxazole yellow
and thiazole orange-based dyes. We expected that the
introduction of dialkylamino group at the 2-position of quinoline
core can generally increase the fluorescence quantum yields
even in the cases of other unsymmetrical cyanine dyes such as
oxazole yellow (YO),[13a] and trimethine dyes such as YO3 and
TO3 which are the core fluorophores of YOPRO3[21] and
TOPRO3[22] dyes, respectively. Therefore, we additionally
synthesized three dyes YONEt2, YO3NEt2, TO3NEt2 bearing
diethylamino group at the 2-positions on quinoline moiety of YO,
YO3 and TO3. While the synthesis of SG and PG remains
unclear albeit their famous names and practical uses, we newly
established the synthetic procedure for a series of dialkylamino-
substituted derivatives of TO and YO (Scheme 1). The synthesis
of monomethine dyes (YONEt2 and TONEt2) began by the
reaction of 2-iodo-1,4-dimethyl-1-quinolinium iodide (1) and
dimethyloxazolinium salt 2[23] or dimethylthiazolinium salt 3[12a]
with triethylamine. After workup with concentrated hydrochloric
acid, the obtained crude chloro-substituted compounds (YOCl
and TOCl) were further reacted with diethylamine to afford
YONEt2 and TONEt2 in 19% and 16% overall yields, respectively.
This synthetic protocol was applicable to the synthesis of
trimethine dyes YO3NEt2 and TO3NEt2 by simply using the two
carbon-extended starting materials 4[24] and 5[24]. Thus, YO3NEt2
and TO3NEt2 were obtained through YO3Cl and TO3Cl in 15%
and 21% overall yields, respectively. For the comparison of
photophysical properties of these dyes, YO, YO3 and TO3,
which do not possess diethylamino group at the 2-position of
quinoline, were also prepared according to the reported
procedures (see SI for details).[24]
R1
X–
N+ R2
R1 = Me, R2 = H, X = TsO: thiaozole orange (TO)
R1 = Me, R2 = NEt2, X = Cl: TONEt2
R1 = Ph, R2 = H, X = Cl: TOPh
S
N
R1 = Ph, R2 = NEt2, X = Cl: TOPhNEt2
thiazole oragne dyes
Figure 2. The structures of TO and newly synthesized derivatives (TONEt2,
TOPh and TOPhNEt2).
Absorption and fluorescent spectra of TO, TOPh, TOPhNEt2,
TONEt2, and commercially available SG were measured in the
absence and presence of dsDNA in tris(hydroxymethyl)-
aminomethane/ethylenediaminetetraacetic acid (Tris-EDTA)
buffer solution, and selected data are shown in Table 1. TOPh,
in which a phenyl group is added on a nitrogen atom of quinoline
dsDNA
substructure of TO, did not affect the ΦF
value (0.22). On
the other hand, TOPhNEt2, in which a diethylamino group was
introduced at the 2-position of quinoline in TOPh, dramatically
enhanced the ΦFdsDNA value (0.76) to the level comparable to SG
(0.74). Interestingly, we found that TONEt2, which has a
diethylamino group on the quinoline but not a phenyl group on a
dsDNA
nitrogen atom, even retained the high ΦF
value (0.79).
These results indicate that the phenyl or methyl group on
nitrogen atom does not affect the quantum yield, and a
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/asia.201601430
Chemistry - An Asian Journal
FULL PAPER
Cl-
I–
i) 2 or 3 or 4 or 5
N+ Cl
N+
I
Et3N, rt
then conc. HCl
X
N
1
O
TsO–
I–
MeS
X
N
Ph
N+
YOCl (n = 1, X = O)
TOCl (n = 1, X = S)
YO3Cl (n = 2, X = O)
TO3Cl (n = 2, X = S)
X
4 (X = O)
5 (X = S)
2 (X = O)
3 (X = S)
N+
Cl-
N+
N
Cl-
N+
N
X
ii) Et2NH
or
CH2Cl2, rt
X
N
N
YONEt2 (X = O): 19%
TONEt2 (X = S): 16%
YO3NEt2 (X = O): 15%
TO3NEt2 (X = S): 21%
Scheme 1. Synthesis of YONEt2, TONEt2, YO3NEt2 and TO3NEt2.
Effect of dialkylamino substitution on photophysical
property of unsymmetrical cyanine dyes. We measured
photophysical properties of newly synthesized dyes (YONEt2,
TONEt2, YO3NEt2 and TO3NEt2) and compared with their
parent dyes without having dialkylamino (diethylamino) group at
the 2-position of quinoline. Figure 3 shows the optical absorption
and fluorescence spectra of YONEt2, TONEt2, YO3NEt2 and
TO3NEt2. Their absorption and fluorescent regions are almost
similar with those of the corresponding mother dye, respectively
(see SI). The dialkylamino-substituted dyes completely cover the
visible region such as blue, green, orange, and red colors. We
summarized the optical properties of these dialkylamino-
substituted dyes in Table 2. Their corresponding mother dyes
YO, TO, YO3 and TO3 are also listed for comparison.
Figure 3. (a) Normalized UV-Vis absorption spectra of dyes possessing
diethylamino group (YONEt2, TONEt2, YO3NEt2 and TO3NEt2) in the
presence of dsDNA in Tris-EDTA buffer solution (pH 8.0). (b) Normalized
steady-state fluorescence spectra excited by corresponding maximum
absorption wavelengths.
We found that the addition of dialkylamino group increased
the quantum yield of dye-dsDNA complexes (ΦFdsDNA). For
To further understand how dialkylamino group affects the
dsDNA
fluorescence
quantum
yield,
fluorescence
lifetime
example, ΦF
of YONEt2 was measured to be 0.90, which is
measurements were performed using time resolved
3 times larger than that of YO (0.30). Because the molar
absorptivity (εdsDNA) of each dialkylamino-substituted dyes is
fluorescence spectroscopy. The fluorescence lifetime of dye-
dsDNA
dsDNA complexes (τFdsDNA) was determined and τF
of
almost similar with that of its corresponding mother dye, the
dsDNA
and εdsDNA
,
YONEt2, TONEt2, YO3NEt2 and TO3NEt2 were 1.5 to 2.4 times
longer than those observed in YO, TO, YO3 and TO3. Because
brightness, which is defined by multiplying of ΦF
become approximately three times larger. It should be also
noted that the increase ratios of the fluorescence intensity by
binding to dsDNA (IdsDNA/Ifree) are comparable with those of the
free
of the extremely low ΦF of these unsymmetrical cyanine dyes,
we mainly focused on the study of dye-dsDNA complexes that
are fluorogenically-activated states.
corresponding mother dye, although the fluorescence quantum
dsDNA
yield of dyes’ free states (ΦFfree) as well as ΦF
also
We calculated the radiative constants (Kr) and non-radiative
dsDNA
dsDNA
constants (Knr) using the τF
and the corresponding ΦF
increases. As our new dyes shine brighter than the previous
unsymmetrical cyanine dyes with comparable fluorescence jump
upon binding to dsDNA, we envisaged that our dyes should stain
dsDNA with high contrast even at the lower excitation-intensity
conditions.
according to following two equations: ΦF = Kr × τF and Kr + Knr
=
–1 [25]
dsDNA
τF
.
The calculated KrdsDNA and Knr
for each dye were
summarized in Table 2. A systematic comparison of the eight
rate constants revealed that dyes possessing a dialkylamino
group showed a large reduction in the Knr values for ca. 1/4 to
1/10-fold and increase in the Kr values for ca. 1.5 to 2.4-fold.
Further scrutinizing how dialkylamino group contributes to the
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/asia.201601430
Chemistry - An Asian Journal
FULL PAPER
changes of each kinetic constant is not an easy task. However, it
can be assumed that the dialkylamino group directly introduced
next to the π-conjugation inhibits the rotation around methine
moiety in the excited state.[26]
Table 2. Summary of photophysical properties.
εdsDNA /10４
Brightness
dsDNA
dsDNA
dsDNA
λabs
/nm [a]
λem
τF
IdsDNA/Ifree [g]
KnrdsDNA /ns
KrdsDNA/ns
dsDNA [d]
free [e]
Compound
ΦF
ΦF
/104 [h]
/nm[c]
/ns [f]
[b]
M-1cm-1
YONEt2
(YO)
467
(486)
5.4
(5.6)
493
(505)
0.90
(0.30)
0.001
(0.0005)
4.3
(3.0)
1900
(1400)
4.9
(1.7)
0.023
(0.23)
0.21
(0.11)
TONEt2
(TO)
488
(509)
6.0
(6.2)
517
(527)
0.79
(0.22)
0.0007
(0.0002)
4.1
(1.7)
4500
(4500)
4.7
(1.4)
0.051
(0.46)
0.19
(0.13)
YO3NEt2
(YO3)
581
(606)
8.5
(8.9)
618
(624)
0.72
(0.17)
0.005
(0.002)
3.7
(2.1)
150
(150)
6.1
(1.5)
0.077
(0.40)
0.20
(0.081)
TO3NEt2
(TO3)
610
(634)
9.5
(9.8)
651
(653)
0.53
(0.15)
0.005
(0.002)
3.4
(1.7)
140
(150)
5.0
(1.5)
0.14
(0.50)
0.16
(0.089)
Tris-EDTA buffer solution (pH 8.0) was used for all optical measurements. [a] Absorption maximum wavelength of dye-dsDNA complexes. [b] Molar absorption
coefficient of dye-dsDNA complexes. [c] Corrected maximum fluorescence wavelength of dye-dsDNA complexes. [d] Absolute fluorescence quantum yield of
dye-dsDNA complexes. [e] Relative fluorescence quantum yield of free dyes. [f] Fluorescence lifetime of dye-dsDNA complexes. [g] Ratio of fluorescence
dsDNA
intensity of dye-dsDNA complex (IdsDNA) over free dye (Ifree). See SI for details. [h] Brightness of dye-dsDNA complexes given by εdsDNA multiplied by ΦF
.
4a). On the other hand, TONEt2 effectively stained nuclei and
nucleoli at the same time (Figure 4b). When ribonuclease
(RNase A) digested cells were used, YONEt2 and TONEt2
clearly stained nuclei and chromosomes with the excellent
fluorescence contrast, suggesting that their fluorescence
become turn-on upon binding to RNA as well as DNA (Figures
4e and 4f). Likely due to their relatively low IdsDNA/Ifree values
(140–150), the two trimethine dyes YO3NEt2 and TO3NEt2 need
ca. 10000-times concentrated conditions (200 µM) to achieve
clear stain of cell nuclei and chromosomes (Figures 4c and 4d).
Although minor change in the staining patterns were observed
when RNase A digested cell was used, the high concentrated
condition is still necessary to achieve the nucleus-selective stain
(Figures 4g and 4h).
Figure 4. Fixed HeLa cell imaging using YONEt2, TONEt2, YO3NEt2 and
TO3NEt2. Cells stained with YONEt2 (a, e) and TONEt2 (b, f) were excited with
a 488 nm laser and their blue and green emission were collected at 490–596
nm. YO3NEt2-stained cells (c, g) were excited with a 560 nm laser and the
orange emission at 560–650 nm was collected. The TO3NEt2-stained cells (d,
h) were excited with a 633 nm laser and red emission at 633–758 nm was
collected. Chromosomes in mitotic cells were indicated by arrowheads. See SI
for detail staining and imaging condition.
Conclusions
Our study revealed that the dialkylamino group at the 2-
position of quinoline is a key component for increasing the
dsDNA
ΦF
values of a series of TO and YO dyes. The newly
dsDNA
synthesized TONEt2 exhibited ΦF
of 0.79 that is higher in
relative to that of commercially available PG.[17] We further
Nucleic acid labeling in fixed HeLa cells imaging with newly
developed unsymmetrical cyanine dyes. Finally, we
evaluated the ability of these newly developed dyes to stain
fixed HeLa cells by using the confocal laser-scanning
microscope (Figure 4). The control staining experiments were
carried out with optimized concentration of 20 nM for YONEt2
and TONEt2, and 200 µM for YO3NEt2 and TO3NEt2. Under
these conditions, YONEt2 strongly stained nucleoli with weak
signals in nuclei, chromosomes and leaving cell cytosol (Figure
extended this finding to other unsymmetrical cyanine dyes such
as YO, YO3, and TO3. The synthesized new dyes YONEt2,
free
TONEt2, YO3NEt2 and TO3NEt2 had very low ΦF
values (<
0.5 × 10-2) in Tris-EDTA buffer solution, but these dyes were
dsDNA
activated to become fluorescent with ΦF
values up to 0.53-
0.90 upon binding with dsDNA. It was also found from the
present systematic study that the introduction of dialkylamino
group is effective to the significantly increase in fluorescent
efficiency (ΦFdsDNA) but not to the turn-on efficiency (IdsDNA/Ifree) of
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/asia.201601430
Chemistry - An Asian Journal
FULL PAPER
[8]
[9]
a) M. R. Gill, J. Garcia-Lara, S. J. Foster, C. Smythe, G. Battaglia, J. A.
Thomas, Nat. Chem. 2009, 1, 662; b) K. E. Erikkila, D. T. Odom, J. K.
Barton, Chem. Rev. 1999, 99, 2777.
unsymmetrical
cyanine
dyes.
Fluorescence
lifetime
measurements supported a critical role of a dialkylamino group
in preventing the non-radiative decay, suggesting that the
rotation around the methine moiety in the excited state can be
prohibited to result in the dramatic increase of ΦF
hope that our present work can lead to the design of new
fluorescence DNA sensitive dyes. For example, by introducing
a) L. G. S. Brooker, A. L. Sklar, H. W. J. Cressman, G. H. Keyes, L. A.
Smith, R. H. Sprague, E. Van Lare, G. Van Zandt, , W. W. Williams, J.
Am. Chem. Soc. 1945, 67, 1875; b) L. G. Lee, C.-H. Chen, L. A. Liu,
Cytometry 1986, 7, 508; c) I. Johnson, M. T. Z. Spence in The
Molecular Probes Handbook, A Guide to Fluoresc ent Probes and
Labeling Technologies, 11th ed., Molecular Probes, Inc., Eugene, OR,
2010, pp. 307.
dsDNA [27]
.
We
dialkylamino group to the 2-positions of quinoline, it may be
possible to increase the ΦF
dsDNA
values of TOPRO and TOTO
[10] a) J. Widengren, P. Schwille, J. Phys. Chem. A, 2000, 104, 6416; b) A.
Sanchez-Galvez, P. Hunt, M. A. Robb, M. Olivucci, T. Vreven, H. B.
Schlegel, J. Am. Chem. Soc. 2000, 122, 2911; c) G. L. Silva, V. Ediz, D.
Yaron, B. A. Armitage, J. Am. Chem. Soc. 2007, 129, 5710.
[11] a) J. R. Carreon, K. M. Stewart, K. P. Mahon Jr., S. Shin, S. O. Kelley,
Bioorg. Med. Chem. Lett. 2007, 17, 5182; b) P. R. Bohländer, H.-A.
Wagenknecht, Org. Biomol. Chem. 2013, 11, 7458.
family dyes. Lastly, we demonstrated that our newly developed
dyes could specifically stain nuclei in fixed HeLa cells with the
confocal laser-scanning microscope. The successful applications
to HeLa cell staining as well as the advantageous features of our
dyes such as relatively smaller molecular weight and high
fluorescence efficiency speak well for the potential of the new
dyes in a range of bioimaging applications.
[12] a) L. Bethge, D. V. Jarikote, O. Seitz, Bioorg. Med. Chem. 2008, 16,
114; b) F. Hövelmann, I. Gaspar, A. Ephrussi, O. Seitz, J. Am. Chem.
Soc. 2013, 135, 19025.
[13] a) H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland,
R. A. Mathies, A. N. Glazer, Nucleic Acids Res. 1992, 20, 2803; b) A. N.
Glazer, H. S. Rye, Nature 1992, 359, 859.
Acknowledgements
This work was supported by the ERATO program from JST (K.I.),
Japan Advanced Plant Science Network, and JSPS KAKENHI
Grant Numbers JP15K14542, JP16H06280, JP16H06465,
JP16H06464. K.U. thanks JSPS for fellowships. Dr. Yasutomo
Segawa is greatly acknowledged for fruitful discussions. ITbM is
supported by the World Premier International Research Center
(WPI) Initiative, Japan.
[14] a) N. Milanovich, M. Suh, R. Jankowiak, G. J. Small, J. M. Hayes, J.
Phys. Chem. 1996, 100, 9181; b) I. Schoen, J. Ries, E. Klotzsch, H.
Ewers, V. Vogel, Nano Lett. 2011, 11, 4008; c) S. Zirkin, S. Fishman, H.
Sharim, Y. Michaeli, J. Don, Y. Ebenstein, J. Am. Chem. Soc. 2014,
136, 7771; d) O. Cavuslar, H. Unal, RSC Adv. 2015, 5, 22380.
[15] a) R. M. Martin, H. Leonhardt, M. C. Cardoso, Cytometry, Part A. 2005,
67, 45; b) T. Suzuki, K. Fujikura, T. Higashiyama, K. Takata, J.
Histochem. Cytochem. 1997, 45, 49.
[16] a) R. W. Sabnis in Handbook of Fluorescent Dyes and Probes, Wiley-
VCH, Weinheim, 2015, p. 408; b) J. R. Lakowicz, G. Piszczek, J. S.
Kang, Anal. Biochem. 2001, 288, 62.
Keywords: cyanine dyes • fluorescence • turn-on DNA probe •
structure-property relationship • HeLa cell imaging
[17] a) V. L. Singer, L. J. Jones, S. T. Yue, R. P. Haugland, Anal. Biochem.
1997, 249, 228; b) G. Cosa, K.-S. Focsaneanu, J. R. N. McLean, J. P.
McNamee, J. C. Scaiano, Photochem. Photobiol. 2001, 73, 585; c) P.
Lebaron, N. Parthuisot, P. Catala, Appl. Environ. Microbiol. 1998, 64,
1725.
[1]
[2]
a) A. Tiselius, Trans. Faraday Soc. 1937, 33, 524; b) J. Sigmon, L. L.
Larcom, Electrophoresis 1996, 17, 1524.
[18] a) Y. Nishimura, T. Higashiyama, L. Suzuki, O. Misumi, T. Kuroiwa,
Euro. J. Cell. Biol. 1998, 77, 124; b) S. Arimura, J. Yamamoto, G. P.
Aida, M. Nakazono, N. Tsutsumi, Proc. Natl. Acad. Sci. USA. 2004, 101,
7805; c) K. Sano-Maeda, S. Sato, T. Ueda, R. Yui, K. Ito, M. Hata, A.
Nakano, K. Murakami-Murofushi, N. Sasaki, Cytologia. 2009, 74, 449.
[19] H. Zipper, H. Brunner, J. Bernhagen, F. Vitzthum, Nucleic Acids Res.
2004, 32, e103.
a) M. Bengtsson, H. J. Karlsson, G. Westman, M. Kubista, Nucleic
Acids Res. 2003, 31, e45; b) R. Higuchi, G. Dollinger, P. S. Walsh, R.
Griffith. Biotechnology, 1992, 10, 413; c) R. Higuchi, C. Fockler, G.
Dollinger, R. Watson. Biotechnology, 1993, 11, 1026.
[3]
[4]
a) C. Riccardi, I. Nicoletti, Nat. Protoc. 2006, 1, 1458; b) G. T. Hirons, J.
J. Fawcett, H. A. Crissman, Cytometry 1994, 15, 129.
[20] J. Nygren, N. Svanvik, M. Kubista, Biopolymers 1998, 46, 39.
[21] C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Nordén, J. Phys.
Chem. 1994, 98, 10313.
G. Lukinavičius, C. Blaukopf, E. Pershagen, A. Schena, L. Reymond, E.
Derivery, M. Gonzalez-Gaitan, E. D’Este, S. W. Hell, D. W. Gerlich, K.
Johnsson, Nat. Commun. 2015, 6, 8497.
[22] K. M. Sovenyhazy, J. A. Bordelon, J. T. Petty, Nucleic Acids Res. 2003,
31, 2561.
[5]
a) Y. Koide, Y. Urano, A. Yatsushige, K. Hanaoka, T. Terai, T. Nagano,
J. Am. Chem. Soc. 2009, 131, 6058; b) J. Eid, A. Fehr, J. Gray, K.
Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman,
A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark,
R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol,
C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y.
Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy,
I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A.
Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A.
Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, S. Turner, Science
2009, 323, 133; c) H. M. T. Choi, V. A. Beck, N. A. Pierce, ACS Nano
2014, 8, 4284.
[23] M. Thompson, Bioconjugate Chem. 2006, 17, 507. S. Laib, S. Seeger,
J. Fluoresc. 2004, 14, 187.
[24] Q. Q. Shi, R. Sun, J. F. Ge, Q. F. Xu, N. J. Li, J. M. Lu, Dyes Pigm.
2012, 93, 1506.
[25] N. J. Turro, J. C. Scaiano, J. C. Scaiano in Principles of Molecular
Photochemistry: An Introduction, University Science Books, Sausalito,
CA, 2009.
[26] F. D. Lewis, J.-S. Yang, J. Am. Chem. Soc. 1997, 119, 3834.
[6]
[7]
J. Kapuscinski, Biotech. Histochem. 1995, 70, 220.
S. A. Latt, J. C. Wohlleb, Chromosoma 1975, 52, 297.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/asia.201601430
Chemistry - An Asian Journal
FULL PAPER
Entry for the Table of Contents
FULL PAPER
Amino magic: A systematic structure-
property relationship study has
uncovered that the dialkylamino group
at the 2-position of quinoline in
unsymmetrical cyanine dyes plays a
critical role in enhancing the
Kakishi Uno, Taeko Sasaki, Nagisa
Sugimoto, Hideto Ito, Taishi Nishihara,
Shinya Hagihara, Tetsuya Higashiyama,
Narie Sasaki, Yoshikatsu Sato,
Kenichiro Itami*
Page No. – Page No.
fluorescence upon binding to double-
strand DNA. The newly developed
dyes were also able to specifically
stain the nucleus in fixed HeLa cells
with a confocal laser-scanning
microscope.
Key Structural Elements of
Unsymmetrical Cyanine Dyes for
Highly Sensitive Fluorescence Turn-
on DNA Probe
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.