Synthesis and evaluation of cyanine-styryl dyes with enhanced photostability for fluorescent DNA staining

Article abstract of DOI:10.1039/c3ob41717d

The photostability of cyanine-styryl dyes of the indole-quinolinium type can be significantly improved by structural variations while the excellent optical properties including the bright fluorescence in the presence of DNA can be maintained or even improved, too.

Full text of DOI:10.1039/c3ob41717d

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Synthesis and evaluation of cyanine–styryl dyes with
enhanced photostability for fluorescent DNA staining†
Cite this: DOI: 10.1039/c3ob41717d
Received 22nd August 2013,
Peggy R. Bohländer and Hans-Achim Wagenknecht*
Accepted 18th September 2013
DOI: 10.1039/c3ob41717d
www.rsc.org/obc
The photostability of cyanine–styryl dyes of the indole– for molecular imaging,14 including the so-called FIT probes
quinolinium type can be significantly improved by structural vari- based on PNA,15 ECHO probes16 and our DNA and RNA “traffic
ations while the excellent optical properties including the bright lights”.17,18 Most recently, a thiazole orange derivative that
fluorescence in the presence of DNA can be maintained or even carries a cyano group at the methane bridge was presented as
improved, too.
a twisted cyanine with superior photostability.19 Alternatively,
dyes of the cyanine–styryl-type represent very promising candi-
dates for molecular imaging of nucleic acids with respect to
their reported property as bright non-covalent binders to
RNA.20 We recently found out that one representative of these
dyes, the cyanine–indole–quinolinium dye (CyIQ), exhibits not
only an excellent brightness in the presence of nucleic acids
but also a photostability that is significantly better than that of
fluorescein, BODIPY and thiazole orange as covalently bound
fluorescent DNA labels.21 Herein, we present the synthesis and
optical spectroscopy of a new set of cyanine–styryl dyes of the
CyIQ type 1–12 with significantly improved brightness, photo-
stability and fluorescence intensity enhancement in the pres-
ence of double-stranded DNA. These dyes represent important
candidates for fluorescent DNA and RNA labelling and mole-
cular imaging inside living cells.
The cyanine–styryl dyes of the CyIQ type consist of a quino-
linium part that is bridged by a double bond to the indole
part. This bridge can be attached either to the 2-position of
the quinolinium moiety (like in 1) or to the 4-position. Accord-
ingly, we divide the discussion of the new synthetic dyes pres-
ented herein into two classes, 1–7 and 8–12 (Scheme 1), and
compare them at the end. All dyes were alkylated by a propyl
linker carrying a terminal hydroxyl group to provide the possi-
bility for the later preparation of the corresponding DNA/RNA
building blocks or for the postsynthetic attachment to DNA or
RNA.22 All synthetic dyes were analytically pure (see ESI†) and
characterized by their absorption and fluorescence (Table 1).
Based on the structure of 1 six new cyanine dyes 2–7
were synthesized with the styryl bridge attached to the 2-posi-
tion of the quinolinium part (Scheme 1). The structures of
these dyes varied: (i) by the N-methyl group at the indole part
(2 vs. 1, 6 vs. 5), by an additional fused benzene ring (3/7 vs. 1)
and by electron-withdrawing/-donating substituents at the
quinolinium part (4/5 vs. 1, 6 vs. 2). First, titration experiments
The synthesis, chemistry and positive evaluation of new
fluorescent dyes that show promising and interesting optical
properties are important prerequisites for applications in
the field of fluorescent bioanalytics and molecular cell
imaging of nucleic acids.1,2 The most important parameters to
evaluate the potential of new dyes are brightness defined as
extinction (ε) × quantum yield (ΦF), photostability and fluo-
rescence intensity enhancement in the presence of nucleic
acids. Especially with respect to the latter property, cyanine
derivatives are in the focus of research since they often exhibit
drastically enhanced fluorescence intensity by binding to DNA
or RNA.3,4 They are also suitable to be assembled as DNA-
templated aggregates.5 Beside fluorescence intensity enhance-
ment, cyanines exhibit high extinction coefficients, generally
good synthetic accessibility, especially optical properties that
can be tuned by organic synthesis, and good quantum
yields.3,4 It is important to mention here that electron-with-
drawing groups, like fluoro substituents and cyano groups, are
able to enhance the photostability of cyanine dyes.6
Thiazole orange probably represents one of the best known
representative dyes of the cyanine class,7 also known as SYBR
Green,8 TO-PRO and in its dimeric form TOTO.9 Its binding
properties as a combination of intercalation and groove
binding4,10 and the fluorescence enhancement in the presence
of DNA has been investigated in detail.11–13 Conjugates of thia-
zole orange with nucleic acids have been prepared and applied
Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT),
Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany. E-mail: Wagenknecht@kit.edu;
Fax: +49-721-608-44825; Tel: +49-721-608-47486
†Electronic supplementary information (ESI) available: Syntheses of dyes 1–12
including images of NMR, MS, IR and elemental analysis, details of titrations,
photostability experiments, quantum yield/extinction coefficient measurements,
analysis of photoproducts. See DOI: 10.1039/c3ob41717d
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bathochromic shift (λshift, ca. 30 nm) of the main UV/Vis
absorption peak of this dye which is a typical observation
upon binding to DNA.23 These absorption changes are: (i) due
to the fact that dye aggregates in aqueous solution are broken
up as a result of DNA binding, and/or (ii) the result of a geo-
metric change (e.g. planarization) of the dye upon binding to
DNA, and/or (iii) the result of the change of polarity in the
DNA binding pocket and/or (iv) due to dipole–dipole inter-
actions between the dye and the DNA bases. According to the
measured fluorescence intensities of dye 5 a saturation plateau
is reached after the addition of approximately 4 equiv. of DNA.
These changes of optical properties have similarly been
observed for all dyes, not only for the ones in this first set 1–7
but also for those in the second set 8–12 that will be described
below. The binding constants were determined (see ESI†) and
lie in the range of 1.5 × 104–4.4 × 104 M−1 (except dyes 7 and 8
which will be discussed below).
In order to elucidate the potential applicability for fluo-
rescent DNA analytics and imaging, Stokes’ shifts (λStokes) and
Scheme 1 Structures of dyes 1–12.
Fig. 1 Representative titration of dye 5 with DNA (10 µM 5, 10 mM Na–Pi
buffer, 250 mM NaCl, 2% ethanol, 0–40 µM dsDNA, λexc = 460 nm), followed by
UV/Vis absorption (top left), change at 451 nm and 480 nm (bottom left), and
by fluorescence (top right), emission intensity enhancement at 558 nm (bottom
were performed with a random sequence double-stranded
DNA. Fig. 1 representatively shows the titration experiment of
dye 5 with DNA which was followed by UV/Vis absorption
and fluorescence spectroscopy. The addition of DNA gives a right). Other titrations are displayed in the ESI.†
Table 1 Optical properties of cyanine dyes 1–12
ε (M−1 cm−1
)
λmax b (nm)
ΦF c (%)
F/F0
λStokes c (cm−1
)
Bc (M−1 cm−1
)
t1/2 e (min)
K (M−1
)
d
Dye
λmax a (nm)
1
2
3
4
5
6
7
8
489
493
487
486
480
481
473
501
509
521
515
522
42 400
42 700
37 700
38 000
39 400
39 300
35 700
26 100
30 800
30 800
27 500
35 700
556
560
555
568
558
556
625
632
590
596
598
602
4.4
6.6
17.0
1.5
5.7
5.8
58
58
99
36
73
46
5
14
34
39
55
59
149
149
147
122
128
133
66
1900
2800
6400
600
2200
1700
2500
3300
3800
5400
3500
5500
90
205
13
1.7 × 104
1.5 × 104
4.3 × 104
1.8 × 104
2.2 × 104
2.0 × 104
2.5 × 105
2.5 × 105
2.9 × 104
3.0 × 104
4.4 × 104
3.9 × 104
50
264
425
130
134
186
331
67
7.1
12.5
12.4
17.6
12.7
15.3
83
9
123
133
120
125
10
11
12
241
a UV/Vis absorption. b Fluorescence. c In the presence of 4.0 equiv. of DNA. d F0: fluorescence intensity without DNA, F: with 3.9 equiv. of DNA.
e In the presence of 2.5 μM DNA.
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below. It is more important to mention here that similar
photooxidation at the indole part (see ESI†) was not obtained
with the N-methylated dyes 2 and 6. Instead, photoproducts
with a ring-opened indole part were observed after longer
irradiation times.24 Obviously, the N-methyl group of 2 and 6
protects the indole part from hydroxylation and thereby from
one of the primary photodegradation pathways at the indole.
Fusing a benzene ring to the quinolinium moiety in dye 3
enhances mainly the quantum yield to 17% due to the
enlarged aromatic system and thereby the brightness of this
dye, but lowers the photostability. On the other hand the
additional benzene ring at the indole moiety of dye 7 (carba-
zole) does not deteriorate the photostability to such an extent,
but yields a quite remarkable Stokes’ shift that is enhanced
from 147–149 cm−1 (1–3) to 66 cm−1 (7) and a significantly
stronger binding to DNA (K = 2.5 × 105 M−1). Obviously the
charge transfer character of the excited state (bonded exci-
plex)25 of this dye is more pronounced.
The work of Armitage et al. has shown that fluoro substitu-
ents at the aromatic parts of cyanine dyes enhance the photo-
stability quite dramatically.6 This has been rationalized by the
electron-withdrawing effect on the conjugated bridge between
the two aromatic parts of the dye that reduces bleaching by
oxygen. Accordingly, dye 4 bears a fluoro substituent (electron-
withdrawing) at the quinolinium part, whereas dyes 5 and 6
carry a methoxy group (electron-donating) at the same posi-
tion. Surprisingly, dyes 5 and 6 but not dye 4 (!) exhibit signifi-
cantly improved photostabilities (by a factor of 3 and 4) and
nearly unchanged optical properties compared to CyIQ (1).
Again, the comparison of the primary photoproducts gives
ESI† for these unexpected results. Hydroxylation of the quinoli-
nium part is a known photochemical degradation26 and has
been observed in 1ox but not 5ox. In the latter compound only
the methoxy substituent has been cleaved to the hydroxyl
group (see ESI†). Obviously, the electron-donating effect or the
photodeprotective reactivity of the methoxy substituent in 5
(and 6) prevents photooxidation of the quinolinium part and
thereby improves photostability of these dyes.
The second set of dyes, 8–12, bear the styryl bridge con-
nected to the 4-position of the quinolinium part. The experi-
ments that were performed to elucidate the optical and
photochemical properties of these dyes were identical to the
ones described above and are summarized in Fig. 3 and again
in Table 1. Remarkably, especially the quantum yields and
photostabilities of dyes 9 and 10 significantly improved in
comparison to the corresponding dyes 1 and 2 in the previous
set (1–7). This can be rationalized by the fact that the 4-posi-
tion of 9 (and 10) is protected by the styryl bridge and thus pre-
vents photooxidation at this position (as evidenced in 1ox).
Photooxidation of 9 is observed at the 2-position (9ox,
Scheme 2) but only after significantly longer irradiation times.
Starting with dye 9 as the regioisomer to 1 we varied the
structure slightly in order to improve the optical properties
and photostabilities. Methylation at the nitrogen of the indole
group yields dye 10 with a remarkable quantum yield and
astonishing photostability. Both values are the best in the
Fig. 2 Photostability of dyes 1–7 measured by the loss of fluorescence intensity
in the presence of DNA (10 µM dye, 2.5 µM dsDNA, 10 mM Na–Pi buffer,
250 mM NaCl, 5% ethanol).
fluorescence intensity enhancements (F/F0) were recorded as
characteristic values for each dye. Quantum yields (ΦF) and
brightnesses (B) were measured in the presence of 4.0 equiv. of
DNA to ensure that the majority of dye molecules are bound to
DNA. The half-life times (t1/2) quantify the photostabilities
during irradiation with a 75 W Xe lamp equipped with a
305 nm cutoff filter to avoid excitation of the DNA com-
ponents. The photodegradation of the dyes was recorded by
the loss of fluorescence intensity in the presence of DNA
(Fig. 2). Table 1 summarizes these values for dyes 1–7.
Dye 2 carries an additional methyl group at the indole nitro-
gen. This small structural change does not alter the optical
properties of the dye significantly but improves the photo-
stability of the dye by a factor of more than 2. A similar result
was obtained by comparison of the methoxysubstituted dyes 5
and 6. In order to study this effect photoirradiated dye
samples were carefully analyzed by HPLC and ESI-MS experi-
ments. In case of the dyes 1 and 5 that are not methylated at
the indole nitrogen the hydroxylated derivatives 1ox and
5ox were identified as primary photooxidation products
(Scheme 2). The UV/Vis absorption of these products indicates
that the conjugation in these uncharged compounds is main-
tained but the fluorescence is lost. Hence, these compounds
must be oxidized at the indole or the quinolinium part. The
hydroxylation at the quinolinium part will be commented
Scheme 2 Photoproducts 1ox, 5ox and 9ox.
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Schwarz (Institute of Physical Chemistry at KIT) for ESI-MS
measurements.
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tried in the set, but an additional benzene ring at the indole
moiety (carbazole) gives dye 8. In comparison to dye 9 (and
similar to dyes 7 vs. 1), the photostability of dye 8 is deteriorated
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all dyes presented herein (K = 2.5 × 105 −1). Finally, two
M
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fluorescent imaging.
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cyanine–styryl dyes of the indole–quinolinium type (CyIQ) can
be improved significantly while the excellent optical properties
including the bright fluorescence in the presence of DNA can
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