Synthesis and study of nucleic acids interactions of novel monomethine cyanine dyes

Article abstract of DOI:10.1016/j.dyepig.2011.08.019

Six asymmetric monomethine cyanine dyes have been synthesized and their spectral characteristics and interaction with double stranded (ds)DNA have been investigated for their prospective use as fluorescent markers in molecular biology. Therefore, the non-covalent binding of the compounds with dsDNA was explored. Apart from the fluorescence spectroscopy, the study includes UV/Vis spectrophotometry and circular dichroism spectroscopy, as well as the thermal melting experiments. Although the compounds show relatively low binding affinity toward dsDNA and do not have intrinsic fluorescence, in the presence of dsDNA they exhibited considerable enhancement in fluorescence intensity. Therefore the studied dyes show interesting platform for future modifications directed toward more sequence selective derivatives. The compound with the highest affinity toward dsDNA showed interesting anti-proliferative potential and specificity.

Full text of DOI:10.1016/j.dyepig.2011.08.019

Dyes and Pigments 92 (2012) 1184e1191
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis and study of nucleic acids interactions of novel monomethine
cyanine dyes
,
a
b
a
Stefka Kaloyanovaa, Ivo Crnolatacb , Nedyalko Lesev , Ivo Piantanida , Todor Deligeorgiev
*
a Department of Applied Organic Chemistry, Faculty of Chemistry, University of Sofia, 1164 Sofia, Bulgaria
b Laboratory for Study of Interactions of Biomacromolecules, Division of Organic Chemistry and Biochemistry, Ruder Boskovic Institute, Bijenicka c. 54, 10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
Six asymmetric monomethine cyanine dyes have been synthesized and their spectral characteristics and
interaction with double stranded (ds)DNA have been investigated for their prospective use as fluorescent
markers in molecular biology. Therefore, the non-covalent binding of the compounds with dsDNA was
explored. Apart from the fluorescence spectroscopy, the study includes UV/Vis spectrophotometry and
circular dichroism spectroscopy, as well as the thermal melting experiments. Although the compounds
show relatively low binding affinity toward dsDNA and do not have intrinsic fluorescence, in the pres-
ence of dsDNA they exhibited considerable enhancement in fluorescence intensity. Therefore the studied
dyes show interesting platform for future modifications directed toward more sequence selective
derivatives. The compound with the highest affinity toward dsDNA showed interesting anti-proliferative
potential and specificity.
Received 16 June 2011
Received in revised form
24 August 2011
Accepted 27 August 2011
Available online 3 September 2011
Keywords:
Cyanine dyes
DNA binding
Fluorescence
Ó 2011 Elsevier Ltd. All rights reserved.
Thermal melting
Circular dichroism
Antiproliferation
1. Introduction
both sides. Therefore, the mode of the DNA binding can vary not
only with the change of the heterocyclic nitrogen subunits or
Although the history of cyanine dyes covers more than century
and a half [1], the interest in their synthesis and various applica-
tions are still present. Their use in molecular biology [2], medical
[3], as well as, clinical analysis [4] has brought them once again into
the limelight. The sensitivity of cyanine based fluorescent markers
for DNA detection and characterization is almost equal to that of
radioactive probes [5], which are less popular because of the safety
concerns. The free cyanines in solution exhibit almost no intrinsic
fluorescence, losing the absorbed energy through the torsional
motion [6] or other nonradiative relaxation, however, upon DNA
binding which results in rigidification of the dye structure, fluo-
rescent intensity enhances significantly. The cyanine dyes are
showing two predominant DNA binding modes: (1) intercalation
between adjacent base pairs or bis-intercalation of some covalently
bridged dimers, and (2) minor groove binding. This dual binding
nature is in accordance with the theoretical observations consid-
ering that their structure implies (poly)methine bridge, which is
flexible and allows twisting, following the curvature of the minor
groove [7], with the planar heterocycles, prone to intercalation, on
multiplicity of the methine bridge but also with the nature, charge
and steric characteristic of the substituents.
2. Materials and methods
2.1. Materials
All starting compounds and solvents were commercial products
from SigmaeAldrich and were used without further purification,
except for compounds 7a [8] and 7d [9], which were synthesized by
modified known procedures.
2.2. Analysis methods and equipment
All products were characterized and/or compared with reported
data. The progress of the reactions was monitored by TLC (Merck F
254 silica gel; dichloromethane: methanol: acetic acid, 86:13:1).
1H-NMR spectra of the compounds that have not been reported
previously were recorded on a Bruker Avance 600 MHz instrument
in DMSO-d6 as solvent. Elemental analyses were performed on
a Vario III instrument. Melting points were determined on a Köfler
apparatus and are uncorrected.
* Corresponding author. Tel.: þ385 14561111/1537; fax: þ385 14680195.
E-mail address: icrnolat@irb.hr (I. Crnolatac).
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2011.08.019

S. Kaloyanova et al. / Dyes and Pigments 92 (2012) 1184e1191
1185
Table 1
1H-NMR, elemental analysis, yields and melting points of dyes aed.
Dye Molecular
formulas
Mw
Yield Melting point
[%]
[ꢀC]
Elemental analysis
1HeNMR, 600 MHz, DMSO-d6,
d (ppm)
Ncalc./found Ccalc./found
Scalc./found
Ocalc./found
a
C25H20BrClN2S. 527.90 71
CH3OH
228e230 decomp. 5.31/5.12
59.15/58.95
6.32/5.94
4.58/4.13 4.25 (s, 3H, NþCH3), 5.77 (s, 2H, CH2), 7.10 (s, 1H, CH),
7.28 (t, 3H, ArH), 7.36 (t, 2H, ArH), 7.57 (d, 1H, ArH),
7.78 (d, 1H, ArH), 7.86 (d, 1H, ArH), 8.51 (d, 1H, ArH),
8.55 (d, 1H, ArH), 8.64 (d, 1H, ArH), 8.68 (d, 1H, ArH),
8.75
(d, 1H, ArH)
b
c
C30H26BrN3O2S. 636.60 60
2CH3OH
245e247 decomp. 6.60/6.32
60.37/60.06
5.38/5.11
5.65/5.47
5.04/4.83 2.16 (m, 2H, CH2), 2.35 (s, 3H, CH3), 3.73 (t, 2H, CH2),
4.25 (s, 3H, NþCH3), 4.51 (t, 2H, CH2), 6.94 (s, 1H, CH),
7.58 (t, 1H, ArH), 7.84 (m, 3H, ArH), 7.87 (m, 2H, ArH),
8.01 (d, 1H, ArH), 8.26 (d, 1H, ArH), 8.35 (s, 1H, ArH),
8.43 (s, 1H, ArH), 8.47 (d, 1H, ArH), 8.50 (d, 1H, ArH)
4.80/4.13 2.36 (s, 3H, CH3), 4.24 (s, 3H, NþCH3), 4.49 (t, 2H, CH2),
4.74 (t, 2H, CH2), 6.93 (t, 1H, ArH), 6.98 (s, 1H, CH),
7.22 (t, 2H, ArH), 7.35 (d, 2H, ArH), 7.58 (t, 1H, ArH),
7.84 (t, 1H, ArH), 8.01 (d, 1H, ArH), 8.10 (d, 1H, ArH),
8.35 (s, 1H, ArH), 8.45 (s, 1H, ArH), 8.47 (d, 1H, ArH),
8.51 (d, 1H, ArH), 9.68 (s, 1H, NH)
C27H25BrN4O2S. 567.50 45
H2O
218e221 decomp. 9.87/10.12 57.14/57.66
d
C24H26ClIN3S2
456.07 63
233e236 decomp. 7.21/6.89
49.45/49.16 11.00/10.84 4.50/4.19 2.10 (s, 4H, CH2SCH2), 2.38e2.42 (m, 4H, CH2CH2),
2.68 (s, 2H, SþCH2), 3.20e3.26 (m, 2H, CH2), 3.50
(s, 3H, CH3), 3.73 (s, 3H, NþCH3), 4.35e4.38
(m, 2H, NCH2), 6.34 (s, 1H, CH), 7.54 (d, 2H, ArH),
7.69 (d, 1H, ArH), 8.02e8.05 (m, 2H, ArH), 8.38
(d, 1H, ArH), 8.48 (d, 1H, ArH), 8.53 (d, 1H, ArH)
2.3. Synthesis of cyanine dyes aef
2.3.1. General procedure for synthesis of dyes aed
N-diisopropylethylamine (0.002 mol) was added and the reac-
tion mixture was stirred at room temperature for 1e4 h.
The progress of the reaction was monitored by TLC. The
In a reaction vessel equipped with magnetic stirrer equimolar
amounts of intermediates 5ae5b (0.001 mol) and 8ae8d
(0.001 mol) were suspended or dissolved in 10 ml ethanol.
resulting precipitate was filtered off, washed with diethyl
ether and air-dried. Dyes aed were recrystallized from
methanol.
O
N
N
O
N
Cl
S
N
H3C
S
N
N
CH3
Br
a
N
CH3
Br
b
HN
O
O
S
N
N
H3C
Br
Cl
S
N
S
N
N
CH3
N
CH3
2I
c
NH
N NH
Cl
I
H3C
I
S
S
N
N
N
CH3
N
CH3
e
f
Scheme 1. Structures of studied compounds.

1186
S. Kaloyanova et al. / Dyes and Pigments 92 (2012) 1184e1191
Scheme 2. Synthesis of intermediates 5a and 5b.
2.3.2. Synthesis of dyes e and f
Dyes e and f were obtained by previously described synthetic
procedure [10].
range lem ¼ 510e650 nm. The titration data were processed using
Scatchard equation [12,13] the resulting values were in the range of
n ¼ 0.3e0.1 however, for the purpose of comparison all Ks values
were re-calculated with the fixed value of n ¼ 0.2. Values for Ks,
given in Table 1, have acceptable correlation coefficients (>0.99).
2.4. Study of DNA/RNA interactions
2.4.2. Thermal denaturation experiments
2.4.1. UV/Visible spectrophotometry, circular dichroism (CD)
and fluorescence spectroscopy
Thermal melting curves for poly dAdTepoly dAdT, poly Aepoly
U and their complexes with studied compounds were determined
as previously described [14] by following the change in the
absorption at 260 nm as a function of temperature. The absorbance
of the ligands was subtracted from each curve and the absorbance
scale was normalized. Tm values are the midpoints of the transition
curves, determined from the maximum of the first derivative and
The UV/Vis spectra were recorded on a Varian Cary 100 Bio
spectrophotometer, fluorescence spectra on a Varian Cary Eclipse
fluorescence spectrophotometer and CD spectra were collected
with a Jasco J-810 spectropolarimeter at 25 ꢀC using 1 cm path
quartz cuvettes.
Absorption spectra to determine the extinction coefficients of
the dyes were obtained on CECIL Aurius 3021 spectrophotometer in
methanol (1 ꢁ 10ꢂ5 mol/l).
checked graphically by the tangent method. The
DTm values were
calculated subtracting Tm of the free nucleic acid from Tm of the
complex. The
D
Tm values (with the instrumental error ꢃ 0.5 ꢀC)
The polynucleotides: poly dAdTepoly dAdT, poly dAepoly dT,
poly dGepoly dC, poly dGdCepoly dGdC, poly Aepoly U, and calf
thymus (ct)-DNA (SigmaeAldrich, St.Louis, USA) were dissolved
in sodium cacodylate buffer, I ¼ 0.05 mol dmꢂ3, pH ¼ 7, calf thymus
reported are the average of at least duplicate measurements.
2.5. Bioassay
(ct-) DNA was additionally sonicated and filtered through a 0.45 mm
filter. Aqueous solutions of compounds were buffered to pH ¼ 7
(sodium cacodylate buffer, I ¼ 0.05 mol ꢁ dmꢂ3). The polynucleotide
concentration was determined spectroscopically [11] as the
concentration of phosphates. Spectrophotometric titrations were
2.5.1. Cell culturing
The experiments were carried out on 2 human cell lines: H460
(lung carcinoma) and HCT 116 (colon carcinoma). Cells were
cultured as monolayers and maintained in Dulbecco’s modified
Eagle medium (DMEM), supplemented with 10% fetal bovine serum
performed at pH ¼ 7.0 (sodium cacodylate buffer, I ¼ 0.05 mol dmꢂ3
)
by adding portions of polynucleotide solution into the solution of
the studied compound. The CD experiments were performed by
adding aliquots of the aqueous solutions of compounds into the
solution of polynucleotide. In fluorimetric experiments the excita-
(FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml
streptomycin in a humidified atmosphere with 5% CO2 at 37 ꢀC.
2.5.2. Proliferation assay
tion wavelength above 500 nm (
the possible inner filter effect caused by increasing absorbance of
the polynucleotide. The emission spectra were collected in the
l
exc > 500 nm) was used to avoid
The panel cell lines were inoculated on day 0 onto a series of
standard 96-well microtiter plates, at 1 ꢁ 104e3 ꢁ 104 cells/ml,
depending on the doubling times of specific cell line. Test agents
Scheme 3. Synthesis of intermediates 8ae8d.

S. Kaloyanova et al. / Dyes and Pigments 92 (2012) 1184e1191
1187
Scheme 4. Synthesis of dyes aed.
were then added in five 10-fold dilutions (from 10ꢂ8 to 10ꢂ4 M)
3. Results and discussion
and incubated for a further 72 h. Working dilutions were freshly
prepared on the day of testing. The solvent was also tested for
possible inhibitory activity by adjusting its concentration to be the
same as in working concentrations. After 72 h of incubation the
cell growth rate was evaluated by the MTT assay [15], which
detects dehydrogenase activity in viable cells. The MTT Cell
Proliferation Assay is a colorimetric assay system, which measures
the reduction of a tetrazolium component (MTT) into an insoluble
formazan product by the mitochondria of viable cells. For this
purpose the medium containing compound was discarded and
medium with MTT was added to each well at a concentration of
3.1. Synthesis and structural analysis of dyes aef
Monomethine cyanine dyes are generally synthesized by the
reaction of two heterocyclic quaternary salts, one bearing a reactive
methyl group and the other a quaternized heterocycle containing
a thioalkyl which is a good leaving group, in the presence of base
[16]. As a part of our previously investigations into monomethine
cyanine dyes, we developed novel method for their synthesis, using
a mixture of a quaternary heterocyclic methylthio salt and non-
quaternized heterocyclic compound bearing a reactive methyl
group in solvent-free conditions and without the presence of
a basic agent [10]. Both methods were used for obtaining of the
dyes aef.
20
mg MTT/40
ml. After 4 h of incubation the precipitates were
dissolved in 160
ml of dymethyl-sulphoxide (DMSO). The absor-
bance (OD, optical density) was measured on a microplate reader
at 570 nm. The absorbance is directly proportional to the cell
viability. The percentage of growth (PG) of the cell lines was
calculated according to one or the other of the following two
expressions:
Intermediates 5a and 5b were prepared according to the re-
ported procedures [17e20] (Scheme 2).
2-Bromoethylphenyl carbamate (7a) was synthesized accord-
ing to modified procedure [8] by refluxing phenylisocyanate and
2-bromoethanol in hexane for 3 h. Sulfonium salt (7d) was
prepared from tetrahydrothiophene and 1,3-diiodopropan in
acetone at room temperature [9]. Intermediates 8ae8d were
synthesized by quaternization of 4-methylquinoline (6) and the
corresponding alkyl or aralkyl halides (7ae7d) under solvent-free
conditions with the corresponding alkyl or aralkyl bromides and
iodides (Scheme 3).
If (mean ODtest ꢂ mean ODtzero) ꢄ 0 then
PG ¼ 100 ꢁ (mean ODtest ꢂ mean ODtzero)/(mean ODctrl ꢂ mean
ODtzero
)
If (mean ODtest ꢂ mean ODtzero) < 0 then:
All intermediates are hygroscopic and their structures were
proved along with the structures of the dyes by 1H-NMR spec-
troscopy and elemental analyses.
PG ¼ 100 ꢁ (mean ODtest ꢂ mean ODtzero)/ODtzero
Where:
Four novel monomethine cyanine dyes were synthesized in
good to high yields and high purity by condensation of quater-
nized 4-methylqunolinium salts (8ae8d) with quaternized
2-methylthioheterocyclic salts (5ae5b) 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.
Mean ODtzero ¼ the average of optical density measurements
before exposure of cells to the test compound.
Mean ODtest ¼ the average of optical density measurements
after the desired period of time.
Mean ODctrl ¼ the average of optical density measurements after
the desired period of time with no exposure of cells to the test
compound.
Each test point was performed in quadruplicate in two indi-
vidual experiments. The results were expressed as GI50, a concen-
tration necessary for 50% of inhibition. Each result is a mean value
from at least two separate experiments.
Dyes e and f were synthesized according to previously reported
novel method [10] (Scheme 5).
Scheme 5. Synthesis of dyes e and f.

1188
S. Kaloyanova et al. / Dyes and Pigments 92 (2012) 1184e1191
d, e, and f were stable showed no significant change in their UV/Vis
spectra over the period of two months. Furthermore, upon heating
up to 90 ꢀC, the UV/Vis spectra of all the studied compounds did not
significantly change, the minor changes (less than 5%) were
reversible after cooling back to room temperature. The UV/Vis
absorption of aqueous solutions of a, b, c, d, e and f at their
respective absorption maxima showed linear correlation to the
concentrations up to c ¼ 2e4 ꢁ 10ꢂ5 mol dmꢂ3, therefore indicating
that there is no significant intermolecular stacking which other-
wise would result in hypochromic shift. Aqueous solutions of a, b, c,
d, e and f at c ¼ 1 ꢁ 10ꢂ6 mol dmꢂ3 (sodium cacodylate buffer,
I ¼ 0.05 mol dmꢂ3, pH ¼ 7.0) exhibited negligible fluorescence upon
excitation at their respective absorbance maxima.
3.3. Study of interactions of a, b, c, d, e and f with DNA and RNA
3.3.1. UV/Vis spectrophotometric titrations
Addition of ct-DNA resulted in batochromic and hypochromic
shifts in UV/Vis spectra of a, b, d (Figs. 2e4), no distinct isosbestic
point could be observed, which suggests mixed binding mode.
Using UV/Vis spectrophotometric data obtained by titration of
compounds with ct-DNA solution, the binding constants Ks and
ratios n[bound compound]/[ct-DNA] were calculated by Scatchard equa-
tion [11,12]. However only with the data acquired by titration of
a and b, the equation gave meaningful results, with the n ratio fixed
at 0.5, compounds a and b showed similar binding constant (log
Ks ¼ 5.41 and 5.39, respectively).
Fig. 1. UV/Vis spectra of compounds a, b, c, d, e and f in methanol (1 ꢁ 10ꢂ5 mol/l).
Table 2
Absorption maxima and extinction coefficients of dyes aef.
Dye
lmax [nm]
3 ]
[l cmꢂ1 molꢂ1
a
b
c
d
e
f
557
550
549
560
549
556
154,600
186,600
131,600
95,000
111,800
94,000
3.3.2. Fluorescence spectroscopy
Although compounds a, b, c, d, e and f did not exhibit consid-
erable intrinsic fluorescence, the fluorescence intensity, compound
solutions increased upon addition of ct-DNA (Fig. 5). The increase of
fluorescence intensity was relatively strong with all the
compounds, except with b, where the increase was quite weak (up
to 30 a.u.). Due to the quite complex pattern of events involved in
fluorescence increase upon binding to DNA the strength of emis-
sion increase could not be directly correlated to the binding affinity.
Strong fluorescence of complexes enabled fluorimetric titrations
and calculation of the binding constants (log Ks) and ratio n[bound
Dyes aef were recrystallized from methanol to obtain the
analytical samples. The structures of the four novel analogs of
Thiazole Orange (aed) were evaluated by 1H-NMR spectroscopy
and elemental analysis (Table 1). Reaction yields and melting points
of the products are summarized in Table 1.
3.2. Physico-chemical properties of aqueous solutions
of studied compounds
that are given in Table 3. Fluorimetric titrations
compound]/[ct-DNA]
The absorption maxima and molar absorptivities of dyes aef
were determined in methanol solutions and are presented in the
Fig. 1 and Table 2.
were performed in buffered (pH ¼ 7.0) aqueous solution, at the
wavelength range (lexc ¼ 550 nm, lemission ¼ 560e700 nm),
significantly distanced from the absorbance range of the ct-DNA
Studied compounds (Scheme 1) are moderately soluble in
redistilled water (about c ¼ 1 ꢁ 10ꢂ4 mol dmꢂ3). According to the
spectrophotometry measurements, the aqueous solutions of a, b, c,
(
l
¼ 220e300 nm).
The majority of the compounds in the study do not show high
binding affinities (Ks values) toward ct-DNA, however the increase
a
b
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.5
0.4
0.3
0.2
0.1
0.0
0.0
5.0x10
1.0x10
c (ct-DNA) / mol dm
1.5x10
2.0x10
2.5x10
400
450
500
550
600
650
/nm
Fig. 2. a) UV/Vis titration of a (c ¼ 2.4 ꢁ 10ꢂ5 mol dmꢂ3) with ct-DNA; b) Changes in UV/Vis spectra of a at lmax ¼ 552 nm upon titration with ct-DNA, at pH ¼ 7, sodium cacodylate
buffer, I ¼ 0.05 mol dmꢂ3
.

S. Kaloyanova et al. / Dyes and Pigments 92 (2012) 1184e1191
1189
0.35
a
b
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.30
0.25
0.20
0.15
450
500
550
600
0.0
5.0x10
1.0x10
1.5x10
/nm
c (ct-DNA) / mol dm
Fig. 3. a) UV/Vis titration of b (c ¼ 2.4 ꢁ 10ꢂ5 mol dmꢂ3) with ct-DNA; b) Changes in UV/Vis spectra of b at lmax ¼ 549 nm upon titration with ct-DNA, at pH ¼ 7, sodium cacodylate
buffer, I ¼ 0.05 mol dmꢂ3
.
a
b
0.60
0.55
0.50
0.45
0.40
0.35
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.00
2.50x10
5.00x10
7.50x10
450
500
550
/nm
600
650
c (ct-DNA) / mol dm
Fig. 4. a) UV/Vis titration of d (c ¼ 3.1 ꢁ 10ꢂ5 mol dmꢂ3) with ct-DNA; b) Changes in UV/Vis spectra of d at lmax ¼ 516 nm upon titration with ct-DNA, at pH ¼ 7, sodium cacodylate
buffer, I ¼ 0.05 mol dmꢂ3
.
Table 3
Stability constants (log Ks),a ratios n ¼ [bound compounds]/[polynucleotide]a and
spectroscopic properties of complexesb of aef with ds-polynucleotides calculated
according to fluorimetric titrations (Na-cacodylate buffer, c ¼ 0.05 mol dmꢂ3, pH ¼ 7.0,
lexc ¼ 550 nm, lem ¼ 560e700 nm, slits 5 nm, c(fcompound) ¼ 1 ꢁ 10ꢂ6 mol dmꢂ3).
of the fluorescence intensity upon ct-DNA binding is considerable.
The exceptions are: b which along with the low Ks value also
exhibits quite low increase of the fluorescence intensity and
d which actually shows relatively high Ks value and not so
log Ks
n
DIcalc
Dl nmc
b
a
b
c
lexc ¼ 550 nm
lexc ¼ 550 nm
lexc¼550 nm
ct-DNA
ct-DNA
ct-DNA
5.28
4.93
5.29
4.94
5.53
5.16
5.06
4.73
6.51
6.03
5.90
5.54
5.82
5.42
5.31
4.97
5.40
5.05
4.94
4.62
fix 0.1
fix 0.2
fix 0.1
fix 0.2
fix 0.1
fix 0.2
fix 0.25
fix 0.5
fix 0.1
fix 0.2
fix 0.25
fix 0.5
fix 0.1
fix 0.2
fix 0.25
fix 0.5
fix 0.1
fix 0.2
fix 0.25
fix 0.5
243
252
27.5
28.3
505
525
529
537
127
142
145
152
327
344
346
352
262
271
273
276
31
a
20
18
400
b
c
d
e
f
300
200
100
0
d
e
f
lexc¼550 nm
lexc¼550 nm
lexc¼550 nm
ct-DNA
ct-DNA
ct-DNA
31
21
31
2.50x10-5 5.00x10-5
c (ct-DNA) / mol dm-3
7.50x10-5
0.00
a
Titration data were processed using Scatchard equation, coefficients of corre-
lation were >0.989e0.999 for all calculated Ks.
b
Emission change; D calc
I
¼ Ilim ꢂ I0; emission intensity change, calculated from
Fig. 5. Changes of fluorescence emission intensity of a, b, c, d, e and f (compound
concentration, c ¼ 1 ꢁ 10ꢂ6 mol dmꢂ3) at their respective maxima upon addition of ct-
DNA at pH 7 (Na-cacodylate buffer, pH ¼ 7.0, I ¼ 0.05 M).
titration data that were processed using Scatchard equation, where I0 is calculated
intensity of examined compound, while Ilim is calculated intensity of complex.
c
Dl, “Stokes shift”, the difference between lexc and lmax emission.

1190
S. Kaloyanova et al. / Dyes and Pigments 92 (2012) 1184e1191
Fig. 6. CD titration of ct-DNA (c ¼ 2.0 ꢁ 10ꢂ5 mol dmꢂ3) with d and f at molar ratios r ¼ [compound]/[polynucleotide] (pH ¼ 7.0, buffer sodium cacodylate, I ¼ 0.05 mol dmꢂ3).
prominent fluorescence increase. However, due to quite complex
pattern of physical events involved in fluorescence increase upon
binding to DNA the strength of emission increase could not be
directly correlated to binding affinity.
result in positive ICD band and intercalation of d should yield
negative ICD band [23], whereby at experimental conditions used
these two ICD effects could neutralize each other, thus yielding
negligible spectral change.
Although compound f induces huge ICD effect with ct-DNA (as
well as with some other synthetic polynucleotides, results not
shown), this happens only at higher concentration ratios r. Such
bisignate ICD signals at higher concentration ratios indicate non-
specific binding along the DNA backbone [23].
3.3.3. Thermal melting experiments
It is well known that upon heating ds-helices of polynucleotides
at well-defined temperature (Tm value) dissociate into two single
stranded polynucleotides. Non-covalent binding of small molecules
to ds-polynucleotides usually has certain effect on the thermal
stability of helices thus giving different Tm values. Difference
between Tm value of free polynucleotide and complex with small
3.4. Proliferation assay
molecule (DTm value) is important factor in characterization of
small molecule/ds-polynucleotide interactions.
Considering the overall low binding affinities and DNA/RNA
stabilization effects of the compounds, only compound d was
checked for its anti-proliferation effect on human cancer cell lines.
The compound showed promising biological activity, exhibiting
order of magnitude higher effect on colon carcinoma (HCT 116,
With the exception of d, the compounds did not stabilize the
double helix of poly dAdTepoly dAdT or poly Aepoly U. The
compound d showed stabilization effect with p(dAedT)2 increasing
with the increase of the concentration ratio r (r ¼ [compound]/
GI50 ¼ 5 ꢃ 2
mM) than toward lung cancer cell line (H460,
[polynucleotide] ¼ 0.1, 0.2 and 0.3;
D
Tm ¼ 2.1, 5.9 and 11.3,
GI50 ¼ 50 ꢃ 35
mM), the specificity which could be of interest.
respectively), while in the case of pApU no effect was observed with
the concentration ratios (r ¼ 0.3) used in the experiment.
4. Conclusions
3.3.4. Circular dichroism (CD) experiments
Six mono and di-cationic unsymmetrical cyanine dyes based on
thiazolopyridinium (aed) and quinolinium (e) or cinnolinium (f)
moieties were synthesized. Four of the dyes are novel (aed). All
except one of them show significant fluorescence increase with the
addition of ct-DNA, which renders their potential in nucleic acid
analysis and detection, however further modification is needed
since majority of the compounds (with the exception of d) show
relatively low binding affinity toward ct-DNA, having 60:40 GC over
AT ratio, and do not stabilize synthetic ATeAT sequence DNA or
AeU sequence RNA against thermal denaturation, therefore
demonstrating ineffective interaction with the polynucleotides.
Along with the absence of the significant changes of the CD spectra
of ct-DNA upon addition of the compounds, the UV/Vis, fluores-
cence spectrophotometric and thermal denaturation assays are all
indicating weak interaction with the DNA/RNA helices. Although,
the compound with the cinnolinium heterocycle, compound f,
showed pronounced induced effect (ICD) on the CD spectra of
ct-DNA and other polynucleotides (poly dAdTepoly dAdT, poly
dAepoly dT, poly dGepoly dC, poly dGdCepoly dGdC and poly
Aepoly U), the character and the intensity of the ICD signal, as
well as the fact that the basepair sequence has no effect on the
occurrence of the ICD signal, points toward non-specific binding.
Introduction of additional cationic charge (compound d) induced
stronger binding, with higher affinity and also resulted in signifi-
cant anti-proliferative effect on human cancer cell lines. In that way
So far, non-covalent interactions at 25 ꢀC were studied by
monitoring the spectroscopic properties of studied compound
upon addition of the polynucleotides. In order to get insight into
the changes of polynucleotide properties induced by small mole-
cule binding, we have chosen CD spectroscopy as a highly sensitive
method toward conformational changes in the secondary structure
of polynucleotides [21] In addition, achiral small molecules can
eventually acquire induced CD spectrum (ICD) upon binding to
polynucleotides, which could give useful information about modes
of interaction [22]. It should be noted that studied compounds are
achiral and therefore do not possess intrinsic CD spectrum.
Except for the compound f addition of all the compounds used
in the study did not result in ICD effect (Fig. 6). The CD response
upon addition of dye d to DNA was located at the wavelength range
where both DNA and dye absorb (220e300 nm), which compli-
cated the deconvolution of effects. However, especially significant
decrease (from ꢂ3 to ꢂ1.5, which makes 50%) of CD intensity at
240e250 nm clearly pointed to significant distortion of DNA
backbone and supported strong binding of d to DNA. The absence
of induced CD (ICD) bands at longer wavelengths where only
d absorbs (>450 nm) could result from several co-existing binding
modes of d to DNA, which is actually in accordance with clear
deviation from the isosbestic points noted in UV/Vis titrations
(Fig. 4). For instance, binding of d into DNA minor groove should

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Ruder Boskovic Institute) for their work on experiments for
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