Synthesis and fluorescence characteristics of novel asymmetric cyanine dyes for DNA detection

Article abstract of DOI:10.1016/j.jphotochem.2010.10.002

Sixteen new asymmetric monomethine cyanine dyes have been synthesized and their spectral characteristics and interaction with double stranded DNA have been investigated. The dyes absorb in the region 453-519 nm and have molar absorptivities in the range 3

Full text of DOI:10.1016/j.jphotochem.2010.10.002

Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Synthesis and fluorescence characteristics of novel asymmetric cyanine
dyes for DNA detection
Stefka Kaloyanovaa,1, Valeriya M. Trusovab,1, Galyna P. Gorbenkob, Todor Deligeorgieva,∗
a Department of Applied Organic Chemistry, Faculty of Chemistry, University of Sofia, 1 James Bourchier Ave., 1164 Sofia, Bulgaria
b Department of Biological and Medical Physics, V.N. Karazin Kharkov National University, 4 Svobody Sq., Kharkov 61077, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen new asymmetric monomethine cyanine dyes have been synthesized and their spectral char-
acteristics and interaction with double stranded DNA have been investigated. The dyes absorb in the
region 453–519 nm and have molar absorptivities in the range 37.900–93.100 l M−1 cm−1. The dyes do
not have intrinsic fluorescence, but in the presence of dsDNA they exhibited a significant enhancement
in fluorescence. The most pronounced increase was found for D9, D10, D12 and D16 allowing the recom-
mendation of these dyes as the most sensitive DNA markers. Thermodynamic analysis of cyanine–DNA
complexation was carried out using the McGhee & von Hippel non-cooperative excluded site model, and
binding parameters have been derived. A hypothesis describing the DNA–dye binding mode has been
proposed.
Received 20 July 2010
Received in revised form
18 September 2010
Accepted 2 October 2010
Available online 29 October 2010
Keywords:
New cyanine dyes
DNA
Binding parameters
Fluorescence
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
which display the light-up effect (strong increase in fluorescence
intensity) when interacting with nucleic acids [8]. Cyanine dyes are
Rapid and highly sensitive quantitative detection of DNA is
crucial in many medical, biological and biotechnological appli-
cations. The most important techniques include the polymerase
chain reaction, DNA sequencing, DNA library development, agarose
gel and capillary electrophoresis staining, DNA damage detection,
purification of DNA fragments for subcloning, the identification
of contaminating DNA in recombinant protein products, flow
cytometry and the evaluation of biological activity [1]. Since the
intrinsic emission of nucleic acids is very weak, extrinsic fluores-
cent probes and labels are generally used for DNA recognition.
A wide range of fluorophores, including ethidium bromide [2],
Hoechst 33258 [3], acridine orange [4], proflavine (acridine-3,6-
diamine) [5], DAPI (4ꢀ,6-diamidino-2-phenylindole) [6] have been
employed to identify DNA [7]. However, these reported molecules
suffer from one severe drawback—they are highly emissive in
solution thereby creating a strong background signal. This disad-
vantage stimulated the search for novel fluorescent probes which
could provide an adequate alternative to the classical fluorphores.
Ideally, a DNA-specific dye should readily switch between none-
missive and emissive states upon binding to nucleic acids. The
most promising dyes with these characteristics are cyanine dyes
photosensitive structures composed of two quaternized, nitrogen-
containing, heterocyclic ring structures, that are linked through
a polymethine bridge [1]. The dyes show large extinction coeffi-
*
cients and a strong ␲–␲ absorption which can be easily tuned
from the visible to the NIR region by synthesizing structural mod-
ifications in the chromophore moiety. This superior property of
cyanine dyes allows them to be programmed in order to achieve
the desired functional fluorescent nanomaterials for DNA visual-
ization. Furthermore, cyanine dyes without intrinsic fluorescence
in solution have a high binding affinity to nucleic acids and illu-
minate only after binding to a substrate. The modification of
benzazole heterocyclic moieties in cyanine dye structures influ-
ences the kind of interaction they have with nucleic acids [9,10].
The substituents at position 6 of the benzothiazole moiety impart
a crescent shape to the dye molecule, particularly the benzoy-
lamino group, thus creating a preference for minor groove-binding
[11].
In attempting to broaden the molecular library of known DNA
reporter molecules, we directed our efforts towards the synthe-
sis and characterization of a range of asymmetric cyanine dyes
differing in their structural features and spectroscopic behaviour.
More specifically, our goals were: (i) to synthesize novel asymmet-
ric analogues of Thiazole Orange with amide substituents in the
benzothiazole moiety; (ii) to identify spectroscopically those fluo-
rophores that would posses the highest sensitivity to dsDNA; (iii)
to describe DNA–cyanine complexation thermodynamically.
∗
Corresponding author. Tel.: +359 2 8161269; fax: +359 2 9625438.
E-mail address: toddel@chem.uni-sofia.bg (T. Deligeorgiev).
1
Both authors contributed equally.
1010-6030/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2010.10.002

148
S. Kaloyanova et al. / Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
2. Materials and methods
cuvette holder (Perkin-Elmer Ltd., Beaconsfield, UK). Excitation
and emission slit widths were set at 10 nm.
2.1. Materials
Fluorescence spectra to determine the fluorescence intensity
of the dye solutions after binding to dsDNA (salmon sperm,
Sigma–Aldrich) were scanned on a Perkin Elmer LS45 fluorescence
spectrometer in 10 mM Tris–HCl, 0.5 mM EDTA buffer, pH 7.4 at
room temperature.
Cattle spleen DNA was obtained from Reakhim (Russia) and
salmon testes DNA was obtained from Sigma–Aldrich. Tris–HCl and
EDTA were obtained from Sigma (St. Louis, MO, USA). All other start-
ing materials and solvents were commercial products of analytical
grade (Sigma–Aldrich or TCI Europe) and were used without further
purification.
2.6. Binding model
Thermodynamic analysis of DNA–cyanine complexation was
made in terms of the McGhee & von Hippel excluded site model
allowing calculation of the binding constant and the stoichiometry
[12]:
2.2. Analysis methods and equipment
1H NMR spectra were recorded on
a Brucker Avance II
ꢂ
ꢃ
600 MHz instrument in DMSO-d6. Elemental analyses were
performed on a Vario III instrument. Melting points were deter-
mined on a Köfler apparatus and are uncorrected. Absorption
spectra were obtained on a Unicam UV 500 spectrophotome-
ter in methanol (1 × 10−5 mol/l). Fluorescence measurements
were taken on PerkinElmer LS45 and LS55 spectrofluorime-
ters.
n−1
ꢀ
ꢁ
B
F
nB
P
1 − (nB/P)
= KP 1 −
(1)
1 − (n − 1)(nB/P)
where B and F are concentrations of bound and unbound dye,
respectively, P is DNA concentration, K association constant, n rep-
resents the site exclusion parameter (i.e. number of DNA base pairs
covered by one dye molecule). The values of K and n were estimated
using the nonlinear least-square fitting procedure.
2.3. Synthesis of cyanine dyes D1-D16
3. Results and discussion
In a reaction vessel equipped with magnetic stirrer equimolar
amounts of intermediates 5a–5d (0.001 mol) and 8a–8g, 10a or 10b
(0.001 mol) were suspended or dissolved in 10 ml ethanol (dyes
D2–D5, D7–D9) or 3 ml acetic anhydride (dyes D1, D6, D10–D16).
N-diisopropylethylamine (0.002 mol) (for D13 0.003 mol) was
added and the reaction mixture was stirred at room tempera-
ture for 1–5 h. The progress of the reaction was monitored by
TLC (Merck F 254 silica gel; dichloromethane:methanol:acetic acid
86:13:1). The resulting precipitate was filtered off, washed with
diethyl ether and air-dried. For compounds D1, D6, D10–D16 when
acetic anhydride was used as reaction media—diethyl ether was
added to the reaction mixture and the precipitated dye was fil-
tered off and air-dried. Dyes D1–D16 were recrystallized from
methanol.
3.1. Synthesis and structural analysis of dyes D1–D16
Monomethine cyanine dyes are generally synthesized by the
reaction of two heterocyclic quaternary salts, bearing respectively
an alkylthio and a methyl group [13].
Intermediates 5a–5d were prepared according to the reported
procedures [14–17] (Scheme 1).
Intermediates 8a–8g and 10a, 10b were synthesized by quater-
nization of 4-methylquinoline (6) and 4-methylpyridine (7) under
solvent-free conditions with the corresponding alkyl and aralkyl
bromides and iodides (Schemes 2 and 3).
All intermediates are hygroscopic and their structures were
proved along with the structures of the dyes by 1H NMR spec-
troscopy and elemental analyses.
The series of novel monomethine cyanine dyes were synthe-
sized in good to high yields and high purity by condensation
of quaternized 4-methylqunolinium or 4-methylpyridinium salts
with quaternized 2-methylthioheterocyclic salts in the presence of
a basic agent (N-diisopropylethylamine) and appropriate solvent,
ethanol or acetic anhydride, at room temperature (Scheme 4). The
dyes obtained were isolated as crude products by direct filtration
in the cases where ethanol was the reaction solvent. When acetic
anhydride was used as reaction solvent the dyes were isolated by
precipitation with diethyl ether followed by filtration.
Dyes D1–D16 were recrystallized from methanol to obtain the
analytical samples. The structures of the novel analogues of Thia-
zole Orange were evaluated by 1H NMR spectroscopy (Table 1) and
elemental analysis (Table 2). Reaction yields and melting points of
the products are summarized in Table 2.
2.4. Preparation of dye and DNA solutions
Stock solutions of dyes were prepared by dissolving the dyes
in ethanol to achieve final concentrations of ∼10 ␮M (1 × 10−5 M).
DNA solutions were prepared in 10 mM Tris–HCl, 0.5 mM EDTA
buffer, pH 7.4. The concentrations of cyanines and nucleic acid
were determined spectrophotometrically using their extinction
coefficients and ε260 = 6.4 × 103 M−1 cm−1 for DNA. The extinction
coefficients of the dyes are given in Table 3.
To determine the fluorescence spectra of dsDNA–dye com-
plexes, appropriate amounts of a stock solution of salmon sperm
dsDNA in 10 mM Tris–HCl, 0.5 mM EDTA buffer, pH 7.4 (TE buffer)
with a concentration 50 ␮g/ml, was added to each dye in TE buffer.
Dye stock solutions with a concentration 1 × 10−4 M were prepared
in DMSO and these were diluted with TE buffer. The final DNA
concentration was 1 × 10−6 M b.p. (8 ␮g/ml) and the final dye con-
centration was 1 × 10−7 M, yielding a molar ratio of dsDNA bp -
dye of 10:1. The mixtures were incubated at room temperature for
15 min prior to spectroscopic measurement.
As seen in Scheme
4 and Table 1, the cyanines under
investigation are asymmetric monomethine dyes, containing a ben-
zothiazole fragment. The dyes are cationic in nature due to the
delocalized positive charge of the chromophore. The dyes display
strong absorption in the range of 453–519 nm and have molar
absorptivities in the range 37.900–93.100 l M−1 cm−1
.
2.5. Fluorescence measurements
Fluorescence measurements were performed in 10 mM
Tris–HCl, 0.5 mM EDTA buffer, pH 7.4 at room temperature using
10-mm path-length quartz cuvettes in a Perkin Elmer LS55 spec-
trofluorimeter equipped with a magnetically stirred, thermostated
3.2. Fluorescence of the free dyes and after binding to dsDNA
Detailed studies of the photo-physical properties of dyes
D1–D16 in the presence of dsDNA (salmon testes) were made

S. Kaloyanova et al. / Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
149
H
H3C
N
H2N
H2N
S
N
S
N
S
N
ii
i
SH
SCH3
SH
O
3
1
2
iii
v
R
H
N
H
N
S
N
H3C
H3C
4b: R = H
S
N
SCH3 4c: R = Cl
4d: R = F
SCH3
SCH3
O
O
O
4b - 4d
4a
iv
iv
R
O
H
N
H
N
S
N
S
N
5b: R = H
SCH3
5c: R = Cl
5d: R = F
CH3SO4
5a
CH3
CH3SO4
CH3
5b - 5d
i : acetone, N-diisopropylethylamine, CH3I, 15 min, r.t., 79% yield [13] ii : (CH3CO)2O, H2SO4, 30 min,
[14a]
[15]
reflux, 89% yield
iii : H2O/NaOH, dimethyl sulfate (DMS), 1.5 h, r.t., 97% yield
iv :
trichloroethylene, DMS, 30 min, reflux, 60-94% yield [16] v : benzoyl chloride (5b) or 2-chlorobenzoyl
chloride (5c) or 2-fluorobenzoyl chloride (5d), EtOH/NaOH, 2 h, r.t., 75% [14b]
Scheme 1. Synthesis of intermediated 5a-5d.
Scheme 2. Synthesis of intermediates 8a–8g.
(Table 3). The dyes do not have any or have only a negligible fluo-
rescence in TE buffer in the absence of dsDNA.
In the presence of dsDNA all dyes show enhanced fluorescence
(Fig. 1). The maximal fluorescence increase was obtained at a
dsDNA concentration of 8 ␮g/ml. Comparison of spectral proper-
ties of free and dsDNA-bound dyes under the same experimental
conditions [18] revealed that the most pronounced fluorescence
increase is observed in D9, D10, D12, D16 (Table 3). The enhance-
ment of cyanine fluorescence in a DNA scaffold is generally
attributed to the hindered torsional rotation around the methane
bridge, which results in the reduction of non-radiative relaxation
of the excited state [1].
3.3. Binding studies
CH3
CH3
120-150oC
30-60 min
To derive the parameters of cyanine–DNA binding, the solutions
of cyanine dyes were titrated with cattle spleen dsDNA (Fig. 2).
The experimental dependencies of dye fluorescence increase
upon binding to DNA (ꢀI) as a function of DNA base pairs-to-dye
molar ratio (P/D) (Fig. 3). These have been analyzed in terms of the
McGhee & von Hippel excluded site model (Eq. (1)). The results
obtained are summarized in Table 4.
The association constants were shown to have magnitudes of
104–105, suggesting that the cyanines examined bind strongly to
DNA. The molar fluorescence (a) and site exclusion parameter,
i.e. number of DNA units occluded by the cyanine molecule, also
have values typical of DNA–dye complexation. Interestingly, the
correlation between variations in fitting parameters (K, n and a)
depending on dye structure was not found. Thus, D12 exhibited
+
X-R1
10a, 10b
N
R1
N
7
X
9
O
(H C)
N
7, 10a :R1 =
X = Br
2
3
O
7, 10b: R1 =
(H2C)3
S
X = I
Scheme 3. Synthesis of intermediates 10a and 10b.

150
S. Kaloyanova et al. / Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
Table 1
Structures, 1H NMR analysis, absorption maxima and extinction coefficients in methanol (1 × 10−5 M) of dyes D1–D16.
Dye structures
1H NMR, 600 MHz, DMSO-d6 ı [ppm]
ꢁmax [nm]
ε [l mol−1 cm−1
]
4.03 (s, 3H, NCH3), 4.56 (t, 2H, NCH2), 4.90 (t, 2H,
CH2O), 6.90 (s, 1H, CH), 6.96 (t, 1H, ArH), 7.23 (t, 2H,
ArH), 7.28 (d, 1H, ArH), 7.36 (s, 2H, ArH), 7.57 (t, 2H,
ArH), 7.63 (t, 1H, ArH), 7.73 (t, 1H, ArH), 7.81 (d, 1H,
ArH), 7.88 (d, 1H, ArH), 7.95–8.00 (m, 3H, ArH), 8.18 (d,
1H, ArH), 8.49 (brs, 1H, ArH), 8.58 (s, 1H, ArH), 8.77 (d,
1H, ArH), 9.71 (s, 1H, NH), 10.59 (s, 1H, NH)
519
70,300
D1
D2
3.73 (s, 3H, NCH3), 4.05 (t, 2H, NCH2), 4.44 (t, 2H,
CH2N), 6.20 (s, 1H, CH), 7.28 (d, 2H, ArH), 7.50–7.63 (m,
4H, ArH), 7.80–7.85 (m, 5H, ArH), 7.99 (d, 2H, ArH),
8.33 (d, 2H, ArH), 8.43 (d, 1H, ArH), 10.54 (s, 1H, NH)
464
37,900
2.06 (t, 2H, CH2), 3.59 (t, 2H, CH2), 3.85 (s, 3H, NCH3),
4.50 (t, 2H, CH2), 6.71 (s, 1H, CH), 7.13 (d, 1H, ArH),
7.37–7.47 (m, 4H, ArH), 7.66–7.73 (m, 6H, ArH), 7.82
(d, 3H, ArH), 8.00 (d, 1H, ArH), 8.42–8.45 (m, 2H, ArH),
8.59 (d, 1H, ArH), 10.44 (s, 1H, NH)
518
81,100
D3
2.10 (s, 3H, CH3CO), 2.22 (t, 2H, CH2), 3.75 (t, 2H,
NCH2), 4.00 (s, 3H, NCH3), 4.66 (t, 2H, CH2N), 6.87 (s,
1H,CH), 7.27 (d, 1H, ArH), 7.65 (dd, 1H, ArH), 7.71–7.77
(m, 2H, ArH), 7.83–7.85 (m, 2H, ArH), 7.87–7.89 (m, 2H,
ArH), 7.97 (t, 1H, ArH), 8.17 (d, 1H, ArH), 8.43 (s, 1H,
ArH), 8.60 (d, 1H, ArH), 8.75 (d, 1H, ArH), 10.32 (s, 1H,
NH)
519
49,900
D4
3.20 (t, 2H, CH2Ph), 4.03 (s, 3H, NCH3), 4.84 (t, 2H,
NCH2), 6.91 (s, 1H, CH), 7.20–7.31 (m, 6H, ArH), 7.58 (t,
2H, ArH), 7.64 (t, 1H, ArH), 7.76 (t, 1H, ArH), 7.82 (d,
1H, ArH), 7.89 (dd, 1H, ArH), 7.99–8.02 (m, 3H, ArH),
8.22 (d, 1H, ArH), 8.32 (d, 1H, ArH), 8.59 (d, 1H, ArH),
8.79 (d, 1H, ArH), 10.60 (s, 1H, NH)
519
77,900
D5
D6
4.12 (s, 3H, NCH3), 5.92 (s, 2H, CH2), 7.03 (s, 1H, CH),
7.36–7.40 (q, 3H, ArH), 7.45 (t, 2H, ArH), 7.49 (d, 1H,
ArH), 7.63 (t, 2H, ArH), 7.70 (t, 1H, ArH), 7.75 (t, 1H,
ArH), 7.93 (t, 2H, ArH), 7.98 (dd, 1H, ArH), 8.01 (d, 1H,
ArH), 8.06 (d, 2H, ArH), 8.70 (d, 1H, ArH), 8.82–8.85 (q,
2H, ArH), 10.69 (s, 1H, NH)
519
73,600

S. Kaloyanova et al. / Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
1H NMR, 600 MHz, DMSO-d6 ı [ppm]
151
Table 1 (Continued)
Dye structures
ꢁmax [nm]
ε [l mol−1 cm−1
]
3.84 (q, 2H, CH2OH), 4.03 (s, 3H, NCH3), 4.67(t, 2H,
NCH2), 5.15 (t, 1H, OH), 6.91 (s, 1H, CH), 7.37 (d, 1H,
ArH), 7.57 (t, 2H, ArH), 7.64 (t, 1H, ArH), 7.75 (t, 1H,
ArH), 7.80 (d, 1H, ArH), 7.89 (dd, 1H, ArH), 7.95–8.01
(m, 3H, ArH), 8.16 (d, 1H, ArH), 8.49 (d, 1H, ArH), 8.58
(s, 1H, ArH), 8.80 (d, 1H, ArH), 10.59 (s, 1H, NH)
516
61,900
D7
2.16 (m, 2H, CH2), 3.69 (t, 2H, NCH2), 3.93 (s, 3H,
NCH3), 4.61 (t, 2H, CH2N), 6.81 (s, 1H, CH), 7.22 (d, 1H,
ArH), 7.44 (t, 1H, ArH), 7.48–7.50 (m, 1H, ArH),
7.54–7.62 (m, 2H, ArH), 7.67 (t, 1H, ArH), 7.71–7.73 (m,
1H, ArH), 7.77–7.82 (m, 5H, ArH), 7.91 (t, 1H, ArH), 8.10
(t, 1H, ArH), 8.52 (d, 1H, ArH), 8.57 (d, 1H, ArH), 8.70 (d,
1H, ArH), 10.84 (s, 1H, NH)
519
64,000
D8
4.04 (s, 3H, NCH3), 4.56 (t, 2H, NCH2), 4.91 (t, 2H,
CH2O), 6.92 (s, 1H, CH), 6.96 (t, 1H, ArH), 7.23 (t, 2H,
ArH), 7.31 (d, 1H, ArH), 7.36–7.40 (m, 4H, ArH),
7.61–7.64 (q, 1H, ArH), 7.71–7.76 (m, 2H, ArH), 7.81 (t,
2H, ArH), 7.98 (t, 1H, ArH), 8.20 (d, 1H, ArH), 7.51–8.56
(m, 2H, ArH), 8.79 (d, 1H, ArH), 9.71 (s, 1H, NH), 10.79
(s, 1H, NH)
519
75,300
D9
4.06 (s, 3H, NCH3), 5.87 (s, 2H, CH2), 6.98 (s, 1H, CH),
7.30–7.34 (m, 2H, ArH), 7.37–7.40 (m, 4H, ArH), 7.43
(d, 1H, ArH), 7.61–7.67 (m, 1H, ArH), 7.70–7.73 (m, 2H,
ArH), 7.82–7.89 (m, 3H, ArH), 7.96 (d, 1H, ArH), 8.61 (s,
1H, ArH), 8.79 (t, 2H, ArH), 10.81 (s, 1H, NH)
519
93,100
D10
3.76 (t, 2H, NCH2), 4.01 (s, 3H, NCH3), 4.66 (t, 2H,
CH2N), 6.87 (s, 1H, CH), 7.28 (d, 1H, ArH), 7.36–7.41 (m,
2H, ArH), 7.62–7.64 (m, 1H, ArH), 7.70–7.74 (m, 2H,
ArH), 7.79 (s, 2H, ArH), 7.83–7.85 (m, 2H, ArH),
7.85–7.88 (m, 2H, ArH), 7.97 (t, 1H, ArH), 8.16 (d, 1H,
ArH), 8.56 (s, 1H, ArH), 8.61 (d, 1H, ArH), 8.75 (d, 1H,
ArH), 10.77 (s, 1H, NH)
518
68,700
D11
D12
4.06 (s, 3H, NCH3), 5.86 (d, 2H, CH2), 6.97 (s, 1H, CH),
7.30–7.35 (m, 3H, ArH), 7.38–7.43 (m, 3H, ArH), 7.51 (t,
1H, ArH), 7.56 (t, 1H, ArH), 7.61–7.70 (m, 3H, ArH),
7.79–7.82 (m, 1H, ArH), 7.86 (q, 2H, ArH), 7.95 (t, 1H,
ArH), 8.62 (d, 1H, ArH), 8.74–8.80 (m, 2H, ArH), 10.91
(s, 1H, NH)
519
85,600

152
S. Kaloyanova et al. / Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
1H NMR, 600 MHz, DMSO-d6 ı [ppm]
Table 1 (Continued)
Dye structures
ꢁmax [nm]
ε [l mol−1 cm−1
]
1.36–1.41 (m, 2H, CH2), 1.54–1.59 (m, 2H, CH2),
1.84–1.88 (m, 2H, CH2), 2.23 (t, 2H, CH2), 4.03 (s, 3H,
NCH3), 4.58 (t, 2H, CH2), 6.92 (s, 1H, CH), 7.35 (d, 1H,
ArH), 7.57 (t, 2H, ArH), 7.64 (t, 1H, ArH), 7.75 (t, 1H,
ArH), 7.82 (d, 1H, ArH), 7.89 (dd, 1H, ArH), 7.97–8.00
(m, 3H, ArH), 8.13 (d, 1H, ArH), 8.59–8.61 (q, 2H, ArH),
8.80(d, 1H, ArH), 10.60 (s, 1H, NH), 12.04 (brs, 1H,
COOH)
517
78,900
D13
2.17 (t, 4H, CH2SCH2), 2.19–2.33 (m, 4H, CH2CH2), 2.62
(t, 2H, S+CH2), 3.21–3.26 (m, 2H, CH2), 3.43–3.53 (m,
3H, CH3CO), 3.73 (s, 3H, NCH3), 4.36–4.39 (q, 2H,
NCH2), 6.35 (d, 1H, CH), 7.52–7.54 (m, 2H, ArH), 7.69
(d, 1H, ArH), 7.38 (d, 1H, ArH), 7.41 (d, 1H, ArH), 8.92
(d, 1H, ArH), 9.84 (s, 1H, NH)
453
66,400
D14
2.10 (s, 4H, CH2SCH2), 2.38–2.42 (m, 4H, CH2CH2), 2.68
(t, 2H, S+CH2), 3.20–3.26 (m, 2H, CH2), 4.36–4.39 (t, 2H,
NCH2), 6.49 (s, 1H, CH), 7.31 (d, 1H, ArH), 7.50 (t, 2H,
ArH), 7.59 (t, 1H, ArH), 7.70 (t, 1H, ArH), 7.81 (d, 1H,
ArH), 7.89 (dd, 1H, ArH), 7.96–8.01 (m, 3H, ArH), 8.12
(d, 1H, ArH), 8.56–8.59 (q, 2H, ArH), 8.79 (d, 1H, ArH),
9.86 (s, 1H, NH)
521
49,200
D15
2.08 (s, 4H, CH2SCH2), 2.35–2.41 (m, 4H, CH2CH2), 2.61
(t, 2H, S+CH2), 3.18–3.22 (m, 2H, CH2), 4.31–4.36 (t, 2H,
NCH2), 6.76 (s, 1H, CH), 7.33–7.39 (m, 2H, ArH), 7.43 (d,
1H, ArH), 7.59–7.61 (m, 4H, ArH), 7.71 (t, 1H, ArH), 7.79
(d, 1H, ArH), 7.96 (d, 1H, ArH), 8.61 (s, 1H, ArH), 8.79 (t,
2H, ArH), 10.81 (s, 1H, NH)
517
58,600
D16
Table 2
Reaction yields, melting points and elemental analysis of the examined dyes.
Dye
Molecular formulae
Mw
Yield [%]
melting point [◦C]
Elemental analysis
N
C
S
H
calc./found
calc./found
calc./found
calc./found
D1
D2
D3
D4
D5
D6
D7
D8
C34H29IN4O3S·1/2CH3OH
C31H25BrN4O3S·1/2CH3OH
C36H29BrN4O3S
C31H27BrN4O3S·CH3OH
C33H28BrN3OS·1/2CH3OH
C32H26BrN3OS·2H2O
C27H24BrN3O2S
716.61
629.54
677.61
647.58
610.58
580.54
534.47
712.05
671.58
598.53
695.60
614.98
604.56
681.43
793.56
717.55
53
83
66
75
41
96
84
46
60
43
43
34
58
75
98
64
180–183
184–186
183–185
178–180
188–190
>300
275–278
175–178
180–182
>300
160–162
>300
145–148
240–243
178–181
160–163
8.00/7.82
9.13/8.90
8.27/8.11
9.10/8.65
7.07/6.88
6.84/6.44
7.86/7.72
7.87/7.55
8.02/7.79
7.02/7.07
7.34/7.38
6.83/7.26
6.51/6.38
5.74/5.29
5.30/5.21
5.86/5.72
58.29/57.82
60.69/60.10
63.81/63.21
60.49/59.35
66.66/65.90
66.20/66.54
60.68/60.46
60.72/60.48
58.45/58.02
64.21/64.36
58.19/58.49
62.50/62.30
59.53/59.02
44.33/45.05
48.43/48.05
53.56/53.05
4.58/4.47
5.23/5.09
4.73/5.33
5.21/4.95
5.39/5.25
5.20/5.68
6.00/6.46
4.50/4.26
4.59/5.79
5.36/5.42
7.34/7.44
5.21/5.62
4.97/4.18
8.77/8.33
8.08/7.78
5.59/5.41
4.17/4.36
4.11/4.32
4.31/5.04
4.42/4.83
4.75/4.95
4.51/3.48
4.53/5.22
3.96/4.23
4.47/5.33
4.21/5.04
4.75/4.79
4.10/3.08
5.46/6.03
4.27/4.61
4.19/3.81
4.50/4.21
C36H28BrClN4O3S
D9
C34H28BrFN4O3S·1(1/2)H2O
D10
D11
D12
D13
D14
D15
D16
C32H25BrFN3OS
C
36H28BrFN4O3S·CH3OH
C32H25BrClN3OS
C31H30BrN3O3S·CH3OH
C23H29I2N3OS2·CH3OH
C32H33I2N3OS2
C32H32Br2FN3OS2

S. Kaloyanova et al. / Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
153
Table 3
Spectroscopic parameters of D1–D16 free in TE buffer and after binding to dsDNA.
Dye
ꢁmax
ꢁmax
ꢁem
Fluorescence intensity of
free dye in TE buffer
Fluorescence intensity
dye + dsDNA
Ratio
TE buffer
TE buffer + dsDNA
D1
D2
D3
D4
D5
D6
D7
D8
507
458
517
518
509
500
503
504
508
501
512
512
509
451
509
510
519
473
530
521
520
520
516
519
519
519
525
519
512
465
512
516
547
496
547
545
546
545
546
543
544
545
545
543
543
496
549
547
2.98
7.91
8.35
1.68
1.31
1.29
2.02
0.89
1.12
0.96
2.09
0.98
2.45
8.79
0.99
0.80
832.51
128.06
446.34
74.66
279
16
53
69
223.52
348.22
695.58
383.41
998.80
992.15
710.18
817.24
941.71
770.15
183.86
349.32
170
270
344
383
892
1033
340
834
385
88
D9
D10
D11
D12
D13
D14
D15
D16
186
437
Table 4
Parameters of cyanine–DNA binding calculated using McGhee & von Hippel model.
D12 free in TE buffer
D12 in presence of dsDNA
Dye
K, ×105, M−1
n
a, ×103, M−1
800
D1
D2
D3
D4
D5
D6
D7
D8
0.66 0.15
2.13 0.63
0.48 0.14
2.37 0.71
0.23 0.07
2.08 0.62
0.47 0.14
1.20 0.36
2.32 0.69
0.53 0.15
0.98 0.29
0.79 0.23
1.19 0.35
7.58 2.27
0.55 0.16
1.27 0.38
2.49 0.74
1.21 0.36
0.16 0.04
1.17 0.35
0.39 0.11
8.37 2.51
0.38 0.11
8.05 2.41
1.29 0.38
1.35 0.40
5.22 1.56
1.31 0.39
10.6 3.1
1.49 0.44
1.95 0.58
1.81 0.54
1.05 0.31
1.51 0.45
1.05 0.31
2.49 0.74
3.24 0.97
2.99 0.89
3.00 0.9
4.79 1.43
3.19 0.95
3.13 0.93
3.19 0.95
1.57 0.47
600
400
200
0
D9
D10
D11
D12
D13
D14
D15
D16
1.78 0.53
3.66 1.09
1.62 0.48
2.56 0.76
540
560
580
600
620
640
660
680
700
wavelength [nm]
Fig. 1. Fluorescence intensity of D12 free in TE buffer and after binding to dsDNA.
cally, the binding constant is determined by molecular interactions
(e.g. hydrophobic, ionic, van der Waals, H-bonding, etc.) stabiliz-
ing complex formation. Molar fluorescence, in turn, is an inherent
spectroscopic characteristic of the dye, proportional to quantum
yield and determined by the ability of the fluorophore to respond
to the changes in its microenvironment [19].
the highest n and a, while D14 has the largest K value. This finding
can be explained by the fact that the association constant reflects
the strength of binding, while the term “molar fluorescence” can
be roughly related to the sensitivity of the dye to DNA. Specifi-
Scheme 4. Synthesis of novel monomethine cyanine dyes D1–D16.

154
S. Kaloyanova et al. / Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
A
120
C
D8
70
60
50
40
30
20
10
0
D15
100
P/D
47.5
P/D
52.5
80
60
40
0
0
20
0
510
510
540
570
600
630
660
690
720
540
570
600
630
660
690
720
Wavelength, nm
Wavelength, nm
450
400
350
300
250
200
150
100
50
B
D
220
200
180
160
140
120
100
80
D16
D14
P/D
42.6
P/D
34
60
0
0
40
20
0
0
520 540 560 580 600 620 640 660 680 700 720
Wavelength, nm
460
480
500
520
540
560
580
600
Wavelength, nm
Fig. 2. Representative fluorescence spectra of cyanine dyes bound to dsDNA.
Below we analyse the molecular events that underlie complex
dsDNA is 2 nm so the space available for intercalation is also about
2 nm. The sizes of dyes D1–D16 were estimated to be 1.8–2.2 nm,
which constitutes an ideal fit for the DNA geometry. Secondly, for
minor groove binding to occur, the dye should possess the degree
of flexibility which is more usual in polymethines [1]. Thirdly,
typical binding constants for organic dye–DNA complexation via
intercalation fall in the range 104–105 M−1, and are usually much
formation between DNA and cyanines. The relative affinity of the
dyes for nucleic acids is largely determined by the nature of their
association. Generally, three main modes of interaction between
guest molecules and DNA occur: (i) intercalation, (ii) minor groove
binding, and (iii) major groove binding. While the latter mode is
specific mainly for large macromolecules such as proteins, inter-
calation and minor groove binding represent the most common
pathways of DNA complexation with small molecules. Intercala-
tion is typical for planar aromatic cationic molecules. This type
of binding results from the incorporation of a dye planar aro-
matic moiety between the DNA base pairs, followed by unwinding
and lengthening of the DNA helix. In contrast, groove binders
are crescent-shaped heteroaromatic structures possessing confor-
mational flexibility which allows the dye molecule to adjust in
the DNA groove. Compared to intercalation, groove binding dis-
torts native DNA conformation to a lesser extent. Unfortunately,
these two modes of dye–DNA interaction cannot be distinguished
by fluorescence spectroscopy. A distinction can only be made if
more sophisticated techniques such as 31P NMR, 1H NMR or cir-
cular and linear dichroism are employed [8]. However, detailed
analysis of the available literature on DNA interactions with flu-
orophores allowed us to suggest that the preferential mode of
cyanine binding to DNA in our case is intercalation. This assumption
was based on the following considerations. Firstly, the geomet-
ric evidence is consistent with this statement. The diameter of
smaller than those observed for groove binders (105–106 M−1
)
[8]. As shown in Table 4, association constants for dyes D1–D16
vary from 2.27 × 104 to 7.58 × 105, lending additional support to
the intercalative binding mode. Finally, for 11 probes the derived
value of n is about 2, the magnitude obeying the “neighbour
exclusion principle” of intercalation which says that the bind-
ing of one intercalating molecule between two DNA base pairs
hinders access of the next binding site to another intercalator
[1,8]. Exceptions are only D9, D11–D14. Site exclusion parame-
ters for these dyes have values ranging from 3 to 4.8. To conclude,
to determine whether it is indicative of another binding mode
or a simple mathematical artefact, additional experiments are
needed.
To summarize, the present work was undertaken to evaluate the
potential of novel asymmetric cyanine dyes to act as non-covalent
DNA labels. The dyes were found to have negligible fluorescence in
the free state, but exhibited a significant emission increase upon
DNA binding. The dyes D9, D10, D12 and D16 showed the most
pronounced fluorescence enhancement. This preferential property

S. Kaloyanova et al. / Journal of Photochemistry and Photobiology A: Chemistry 217 (2011) 147–156
155
90
80
70
60
50
40
30
20
10
0
A
D
D8
70
D9
60
D3
D13
D11
50
D1
40
30
20
10
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
P/D
P/D
20
18
16
14
12
10
8
B
E
D4
240
210
180
150
120
90
D12
D7
D6
D2
6
D15
60
4
30
2
0
0
0
20
40
60
80
0
5
10 15 20 25 30 35 40 45 50 55 60 65 70
P/D
P/D
400
360
320
280
240
200
160
120
80
C
F
400
350
300
250
200
150
100
50
D14
D10
D5
D16
40
0
0
0
50
100
150
200
250
300
0
5
10
15
20
25
30
35
40
P/D
P/D
Fig. 3. Isotherms of binding of cyanine dyes D1–D16 to dsDNA.
allows us to recommend these dyes as the most sensitive DNA
probes.
Fundamental Research State Fund. VT gratefully acknowledges an
award by Human Frontier Science Program.
Acknowledgements
References
This work was supported by the grant no. 4534 from the Science
and Technology Center in Ukraine and grant no. F28.4/007 from
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