Synthesis and spectroscopic characterisation of (E)-2-(2-(9-(4-(1H-1,2,4- triazol-1-yl)butyl)-9H-carbazol-3-yl)vinyl)-3-ethylbenzo[d]thiazol-3-ium, a new ligand and potential DNA intercalator

Article abstract of DOI:10.2478/s11696-013-0343-7

Three new compounds based on carbazole planar skeleton were synthesised. Among them there is a new ligand and a potential DNA intercalator which contains a benzothiazolium moiety connected to the carbazole ring by a vinyl bridge. The absorption and emission spectral properties of this new ligand have been studied by spectroscopic methods.

Full text of DOI:10.2478/s11696-013-0343-7

Chemical Papers 67 (9) 1231–1239 (2013)
DOI: 10.2478/s11696-013-0343-7
ORIGINAL PAPER
Synthesis and spectroscopic characterisation
of ( )-2-(2-(9-(4-(1 -1,2,4-triazol-1-yl)butyl)-9 -carbazol-3-
yl)vinyl)-3-ethylbenzo[ ]thiazol-3-ium, a new ligand
and potential DNA intercalator‡
Agata Glꢀuszyn´ska*, Ewa Rajczak, Bernard Juskowiak
Laboratory of Bioanalytical Chemistry, Faculty of Chemistry, A. Mickiewicz University,
Umultowska Str. 89b, 61-614 Poznan´, Poland
Received 15 June 2012; Revised 22 November 2012; Accepted 24 November 2012
Three new compounds based on carbazole planar skeleton were synthesised. Among them there is
a new ligand and a potential DNA intercalator which contains a benzothiazolium moiety connected
to the carbazole ring by a vinyl bridge. The absorption and emission spectral properties of this new
ligand have been studied by spectroscopic methods.
c
ꢀ 2013 Institute of Chemistry, Slovak Academy of Sciences
Keywords: carbazole derivative, UV-VIS absorption spectra, fluorescence, G-quadruplexes, DNA
Introduction
Sun et al. (1997), the number and diversity of “G-
quadruplex ligands” with potential anticancer prop-
erties has grown rapidly (Riou, 2004; Kelland, 2005;
De Cian et al., 2008; Folini et al., 2009; Brooks &
Hurley, 2010). Among them, one derivative of car-
bazole, 3,6-bis[2-(1-methylpyridinium)vinyl]carbazole
(BMVC), can recognise specific quadruplex struc-
tures, particularly the quadruplex of the human telom-
eric sequence d(T2AG3)4, and can thus inhibit telom-
erase (IC50 = 5 × 10−8 M) (Chang et al., 2003a,
2003b, 2004a, 2004b, 2006a, 2007; Chang & Chang,
2010; Tsaia et al., 2007; Yang et al., 2007; Huang et
al., 2008).
Carbazole derivatives are interesting also because
of their other biological and photophysical properties
(Chang et al., 2006a, 2006b; Li et al., 2009; Patrick et
al., 1997; Spychalꢀa, 2009; Tanious et al., 2001; Dias et
al., 2004; Hotzel et al., 2002; Saengkhae et al., 2007;
Glꢀuszyn´ska et al., 2010, 2012; Dumat et al., 2011; Qu
et al., 2008). For example, carbazole dications repre-
sent an unusual class of DNA binding agents that are
active against opportunistic infections (Patrick et al.,
Small molecules can noncovalently interact with
DNA via intercalation or minor groove binding. The
majority of classical DNA ligands have been addressed
to the applications with double stranded DNA. In
recent years, interest in ligands capable of interact-
ing with the triple stranded DNA helix and stabilis-
ing G-quadruplexes has increased significantly (Ker-
win, 2000; Cuesta et al., 2003; Davis, 2004; Riou,
2004; White et al., 2007; De Cian et al., 2008;
Ou et al., 2008; Franceschin, 2009; Jain & Bhat-
tacharya, 2011). G-quadruplexes are unusual struc-
tural forms of nucleic acids, first reported by Gellert
et al. (1962). The G-rich nucleic acid sequences are
able to form of G-quadruplex conformation (contain-
ing flat G-quartets) which consist of four guanines held
together by eight hydrogen bonds. These tetraplex
structures are stabilised in the presence of specific
metal cations (Na+, K+) or small organic ligands. The
first report on biological activity of G-quadruplexes
(inhibition of human telomerase) was published by
*Corresponding author, e-mail: aglusz@amu.edu.pl
‡
Presented at the 39th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare,
21–25 May 2012.

1232
A. Gꢀluszyn´ska et al./Chemical Papers 67 (9) 1231–1239 (2013)
1997). Some derivatives of carbazole with antibacte-
rial activity behave essentially as DNA minor groove
binders (Patrick et al., 1997; Spychalꢀa, 2009; Tanious
et al., 2000, 2001) although they contain a planar
chromophore which is generally a feature characteris-
tic of DNA intercalators. The compounds of carbazole
structure also inhibit topoisomerase I and II (Patrick
et al., 1997; Dias et al., 2004; Hotzel et al., 2002).
Several classes of drugs have been reported to inhibit
β-amyloid fibril formation, carbazoles included. Inhi-
bition of this peptide accumulation, characteristic of
the Alzheimer disease, may be a method of prevention
of this disease (Saengkhae et al., 2007).
22Htel) were purchased from Sigma–Aldrich (Poznan´,
Poland) and Genomed (Warszawa, Poland), respec-
tively, and were used without further purification. The
tetramolecular parallel G-quadruplex was prepared by
heating to 95◦C for 10 min and slowly cooling to
room temperature and then by equilibrating at 4◦C
for 24 h before use. Concentrations of ctDNA and
G4 DNA were determined by UV absorbance mea-
surements using the extinction coefficient calculated
at 260 nm and 85◦C. All spectrophotometric titration
experiments were conducted under controlled condi-
tions (in dark) to avoid any photoisomerisationof the
dye during titration. Titrations were made in a Tris-
HCl buffer (10 mM, pH 7.2) in the presence of 100 mM
NaCl or 150 mM KCl solutions. A typical titration
consisted of a successive addition of small amounts of
concentrated DNA solution to a 10.9 µM (ctDNA) or
8.03 µM (G4 DNA) solution of the dye, followed by
stirring, thermal equilibration, and recording of the
spectrum.
Experimental
Melting points were determined on a Koffler block
and are not corrected. IR spectra (in KBr pellets)
were obtained with an FT Bruker IFS 66v/S instru-
1
ment. H (400 MHz) NMR spectra were recorded on
a Varian Gemini 400 using TMS as the internal stan-
dard. Mass spectra (EI) were measured on an AMD-
402 with the acceleration voltage of 8 kV, electron
energy of 70 eV and ion source temperature of 200◦C;
LR-1000, HR-5000. Analytical HPLC was performed
on a LaChrom HPLC system (Hitachi) consisting of
an L-7300 column oven, a dual L-7100 pump system,
an L-7450H diode array detector, and a D-7000 HPLC
interface on an InertSil ODS 3 column, (dp = 5 µm,
250 mm × 4.6 mm i.d. (G1 Science)) with isocratic
elution (eluent: MeOH/10 mM NaCl (ϕr = 7 : 3); flow
rate of 1 mL min−1). An HPLC–MS system (Waters)
type 2690 consisting of a photodiode array detector
type 2996, a column (dp = 5 µm, 150 mm × 4.6 mm
i.d.) with isocratic elution (the eluent: MeOH/H2O
(ϕr = 1 : 1); flow rate 0.5 mL min−1), and a mass chro-
matograph Micromass & Waters. Merck DC-Alufolien
Kieselgel 60254 were used for TLC. Filtered water
(POLWATER D-100 UIM) was used in all experi-
ments. A solution of ligand VI was prepared by dis-
solving a solid sample in DMSO. The absorption spec-
tra were recorded on a Cary-100 spectrophotometer
equipped with a temperature controller. The cell com-
partments were thermostated at 25◦C. Absorption
temperature spectra of the ligand were recorded in
the range of 25–90◦C. Fluorescence spectra (in the
range of 400–700 nm with both excitation and emis-
sion slits of 1 nm) were taken on a spectrofluorime-
ter Jasco FP 6200. The irradiation experiment pro-
ceeded as follows: a dye solution (1.53 × 10−5 M) in
a 10 mM Tris-HCl buffer was irradiated in the spec-
trofluorimeter Cary Eclipse equipped with a pulsed
xenon lamp with excitation and emission band widths
of 20 nm and 5 nm, respectively, at 451 nm (1 h) and
then at 272 nm (1 h). All measurements were carried
out using a 10 mm quartz cell. Double stranded Calf
thymus DNA (ctDNA) and synthetic polynucleotide
5ꢀ-AGGGTTAGGGTTAGGGTTAGGG-3ꢀ (G4 DNA,
9-[4-(1H-1,2,4-Triazol-1-yl)butyl]-9H-carbazole-3-
carbaldehyde (IV) and 9-(4-(1H-1,2,4-triazol-1-yl)
butyl)-9H-carbazole-3,6-dicarbaldehyde (V) were syn-
thesised according to already published methods
(Zhang et al., 1996; Kim et al., 1998; Xu et al., 2006).
(E)-2-(2-(9-(4-(1H-1,2,4-Triazol-1-yl)butyl)-
9H-carbazol-3-yl)vinyl)-3-methylbenzo[d]
thiazol-3-ium (VI)
3-Ethyl-2-methyl-1,3-benzothiazolium iodide (25.9
mg, 0.085 mmol) was added to a solution of 9-[4-(1H-
1,2,4-triazol-1-yl)butyl]-9H-carbazole-3-carbaldehyde
(IV ) (27 mg, 0.085 mmol) in MeOH (1.5 mL). The re-
action mixture was protected from light. After 15 min,
piperidine (4.203 µL, 0.042 mmol) was added drop-
wise to the yellow-orange solution. The reddish solu-
tion was stirred at ambient temperature for 24.5 h.
The reaction progress was monitored by TLC using
CHCl3/MeOH/NH4OH (ϕr = 9 : 0.9 : 0.1) as the elu-
ent. Compound VI was obtained as pure (by HPLC),
intensely orange solid in a 92 % yield.
Results and discussion
Our research group is interested in the synthesis of
ligands, small molecules based on carbazole skeleton,
with interesting biological and photophysical proper-
ties which can bind to secondary structures of DNA.
The new prepared ligand, a potential DNA intercala-
tor, shows the following features: (i) it has a carbazole
planar rigid structure containing three condensed aro-
matic rings, which means that the ligand should show
the ability to interact with DNA (Patrick et al., 1997;
Tanious et al., 2001; Cuesta et al., 2003; Chang et al.,
2003a, 2003b, 2004a, 2004b; Dias et al., 2004; Zhang
et al., 2010); (ii) it has a benzothiazolium moiety;
the presence of a cationic group enhances the ligand’s

A. Gꢀluszyn´ska et al./Chemical Papers 67 (9) 1231–1239 (2013)
1233
Fig. 1. Reagents and conditions: i) Br(CH2)4Br, K2CO3, acetone, 90◦C, 23 h; ii) 1H-1,2,4-triazole, K2CO3, tetrabutylammonium
bromide (TBAB), CH3CN, 45◦C (1 h), 80◦C (25 h); iii) POCl3, DMF, Cl(CH2)2Cl, 25◦C (2 h), 90◦C (22 h); yield: 49 % of
IV, 26 % of V; iv) POCl3, DMF, Cl(CH2)2Cl, 90◦C, 6 h; yield: 50 %; v) 3-ethyl-2-methyl-benzothiazolium iodide, MeOH,
piperidine, 25◦C, 24.5 h.
solubility in aqueous environment and allows electro-
static interactions with DNA; benzothiazolium moiety
is known as a structural element of highly fluorescent
cyanine dyes (Haugland, 1996); (iii) it has a double
bond enabling the control of the ligand structure by
light (cis/trans photoisomerisation) (Juskowiak et al.,
1999, 2002).
type condensation between monoaldehyde IV and 3-
ethyl-2-methylbenzothiazolium iodide in MeOH using
piperidine as the base. It was characterised by a pos-
itive mode m/z 478 [M]+ and a negative mode m/z
1
127 [M]− peaks in the ES-MS, and by H NMR spec-
tral data. The E configuration of VI was established
on the basis of a high value of the coupling constant
—
Herein, the synthesis of a (E)-2-(2-(9-(4-(1H-1,2,4-
triazol-1-yl)butyl)-9H-carbazol-3-yl)vinyl)-3-ethyl-
benzo[d]thiazol-3-ium ligand (VI ), expected to inter-
act with G-quadruplex (Fig. 1), is reported. The start-
ing 9-N-[4-(1H-1,2,4-triazol-1-yl)butyl-carbazole (III )
was prepared from carbazole by a known synthetic
procedure in a 30 % overall yield (Zhang et al., 2010).
From among the known synthetic procedures of the
Vilsmeier formylation of carbazole compounds (Zhang
et al., 1996; Kim, 1998; Xu et al., 2006; Ryu, 2006;
Song et al., 2008), three were chosen, differing in the
amount of reagents used and in the reaction condi-
tions.
The use of a large excess of reagents resulted in a
mixture of aldehydes IV and V with moderate yields,
49 % and 26 %, respectively (Zhang et al., 1996; Kim,
1998). The use of other reaction conditions resulted
only in mono derivative IV with a 50 % yield (Xu et
al., 2006).
J = 15.5 Hz for protons of the CH CH double bond,
—
found as an isolated doublet at δ 7.78 and a doublet
in the group of aromatic protons at δ 8.03. The purity
of VI was checked by HPLC.
The newly synthesised ligand VI is soluble and sta-
ble in aqueous solutions (EtOH/H2O, DMSO/H2O),
selected organic solvents, and in the Tris-HCl buffer.
The UV-VIS absorption spectra (Fig. 2) with a maxi-
mum absorption band at about 480 nm are charac-
—
—
teristic of a CH CH double bond connecting car-
bazole and benzothiazolium moieties, and the exact
location of the absorption maximum is correlated with
the polarity of the solvents (shift up to 75 nm) as
shown in Fig. 3 and Table 1. In moderately polar sol-
vents such as CH2Cl2 and CHCl3, absorption bands
are red-shifted (502–507 nm) compared with those in
polar solvents. Moreover, the intensity of the absorp-
tion bands is higher in these solvents (Fig. 2, Ta-
ble 2). The absorption bands in polar solvents such
as MeOH, EtOH, and CH3CN, are shifted to the
Compound VI was prepared by the Knoevenagel

1234
A. Gꢀluszyn´ska et al./Chemical Papers 67 (9) 1231–1239 (2013)
Table 1. Characterisation and spectral data of newly prepared compounds
Compound
Spectral data
IV
M.p.: 106–110◦C
IR, ν˜/cm−1: 2855, 2802 (C—H), 1684 (C O)
—
—
1H NMR (CDCl3), δ: 1.90–2.00 (m, 4H, CH2CH2CH2CH2), 4.12 (t, J = 6.4 Hz, 2H, CH2—triazole), 4.40 (t, J =
6.6 Hz, 2H, CH2—carbazole), 7.33–7.37 (m, 1H, ArH), 7.42–7.47 (m, 2H, ArH), 7.52–7.56 (m, 1H, ArH), 7.93 (s,
2H, H-3 and H-5 of triazole), 8.00–8.03 (m, 1H, ArH), 8.16–8.18 (m, 1H, ArH), 8.63 (s, 1H, ArH), 10.11 (s, 1H,
—
CH O)
—
ES-MS (positive mode), m/z (Ir/%): 319 ([M + H]+, 100), 341 ([M + Na]+, 24)
EI-MS, m/z (Ir/%): 319 (M+ + 1, 16), 318 (M+, 61), 208 (100)
HRMS, m/z (found/calc.): 318.14823/318.14807 ([M + H]+, C19H18N4O)
V
M.p.: 157–159◦C
IR, ν˜/cm−1: 2805, 2721 (C—H), 1684 (C O)
—
—
1H NMR (CDCl3), δ: 1.71–2.04 (m, 4H, CH2CH2CH2CH2), 4.18 (t, J = 6.3 Hz, 2H, CH2—triazole), 4.21–4.55
(m, 2H, CH2—carbazole), 7.52–7.54 (m, 2H, ArH), 7.94 (s, H-1 and H-5 of triazole), 7.99 (s, 1H, H-3 of triazole),
—
8.07–8.10 (m, 2H, ArH), 8.66 (s, 2H, H-4 and H-5 of carbazole), 10.13 (s, 1H, CH O)
—
EI-MS m/z (Ir/%): 347 (M+ + 1, 20), 346 (M+, 79), 236 (100)
HRMS, m/z (found/calc.): 346.14304/346.14297 ([M + H]+, C20H18N4O2)
VI
M.p.: 252–255◦C
IR, ν˜/cm−1: 1608 (C N), 958 (CH CH)
—
—
—
—
1H NMR (DMSO-d6), δ: 1.52 (t, J = 7.2 Hz, 3H, NCH2CH3), 1.75 (t, J = 7.2 Hz, 2H, CH2CH2—triazole), 1.85
(t, J = 7.1 Hz, 2H, CH2CH2—carbazole), 4.22 (t, J = 6.8 Hz, 2H, CH2—triazole), 4.53 (t, J = 6.5 Hz, 2H, CH2—
carbazole), 4.97–5.00 (m, 2H, NCH2CH3), 7.35 (t, J = 7.7 Hz, 1H, ArH), 7.56 (t, J = 7.5 Hz, 1H, ArH), 7.78 (d, J =
—
—
15.4 Hz, 1H, CH CH), 7.72–7.89 (m, 4H, ArH), 7.94 (s, 1H, ArH), 8.03 (d, J = 15.5 Hz, 1H, CH CH), 8.23–8.28
—
—
(m, 3H, ArH), 8.42–8.48 (m, 3H, ArH), 8.93 (s, 1H, ArH)
ES-MS, m/z (Ir/%): 478 (M+, 100), 127 (M−, 100)
Fig. 3. Normalised long-wavelength UV-VIS absorption band
of VI (2.94 × 10−5 M) in selected solvents and in aque-
ous solutions: toluene (1), Tris-HCl buffer (2), H2O (3),
5 vol. % aq. EtOH (4), 5 vol. % aq. DMSO (5), CH3CN
(6), DMSO (7), MeOH (8), EtOH (9), 1,4-dioxane (10),
CH2Cl2 (11), CHCl3 (12).
Fig. 2. Absorption spectra of VI (2.94 × 10−5 M) in se-
lected solvents and in aqueous solutions: toluene (1),
1,4-dioxane (2), Tris-HCl buffer (3), 5 vol. % aq. EtOH
(4), DMSO (5), CH3CN (6), EtOH (7), CHCl3 (8).
shorter wavelengths (470–479 nm) and their intensi-
ties are slightly lower. In the aqueous Tris-HCl buffer,
and aqueous mixed solutions containing 5 vol. % of
EtOH or DMSO, the absorption spectra are even more
blue-shifted (451–454 nm) and much less intense. Only
the absorption band of the ligand solution in toluene
is of much lower intensity and clearly blue-shifted
(432 nm), which suggests the formation of dimers or
higher aggregates. Such phenomena are usually mani-
fested by changes in the absorption spectrum and the
appearance of fluorescence quenching or excimer emis-
sion in the fluorescence spectrum.
Aqueous solutions (water, 5 vol. % EtOH, 5 vol. %
DMSO, Tris-HCl buffer) of VI designed as potential
DNA binding agent are stable in time. No aggregation
or precipitation phenomena were observed for these
solutions. Spectra were recorded after 5 min and 10
min after every addition of the ligand and then every
half an hour (no addition). These spectra overlapped
and were practically indistinguishable (Fig. 4).
The existence of a monomeric form of the ligand
was also confirmed by the changes in the absorption

A. Gꢀluszyn´ska et al./Chemical Papers 67 (9) 1231–1239 (2013)
1235
Table 2. Effect of solvent on the spectral characteristics of the absorption and fluorescence spectra of VI (concentration:
2.94 × 10−5 M and 1.36 × 10−6 M for the absorption and fluorescence spectra, respectively)
Absorption spectra
Fluorescence spectra
Solvent
λmax/nm
ε · 10−4/(mol−1 cm−1
)
λmax/nm
λmax/nm
CHCl3
CH2Cl2
MeOH
CH3CN
507
502
474
470
479
454
453
452
472
451
482
432
5.31
5.25
5.05
4.87
4.81
4.34
4.29
4.21
4.16
4.07
2.35
2.09
508
508
474
464
482
461
464
464
460
472
486
nd
568
563
568
570
568
565
565
563
564
573
567
nd
EtOH
5 vol. % aq. DMSO
5 vol. % aq. EtOH
H2O
DMSO
Tris-HCl buffer
1,4-Dioxane
Toluene
Note: nd – not detected.
Fig. 4. Stability of VI solutions in the Tris-HCl buffer. Spec-
tra measured after 5 min and 10 min after an addition
of every 4 µL (2.99 × 10−6 M) of the ligand. [VI]:
2.99 µM (1), 5.98 µM (2), 8.94 µM (3), 11.9 µM (4),
14.85 µM (5), 17.79 µM (6), 20.71 µM (7), 23.62 µM
(8), 26.52 µM (9), 29.41 µM (10) and then every half
an hour (0–3 h) (11–16).
spectra in the range of 25–90◦C (Figs. 5a and 5b). The
shape of the spectra did not change, only a small shift
towards longer wavelengths was observed (∆λ = 5 nm)
(Fig. 5b). The temperature stability of the ligand is
important for further studies with DNA.
The fluorescence spectra of VI were recorded in
different solvents and aqueous solutions as shown in
Fig. 6. Significant differences in the intensity of fluo-
rescence bands and small shifts in the emission max-
ima, λem (563–573 nm), were observed. The fluores-
cence spectrum of VI exhibits a broad weak emission
band at 563–565 nm in aqueous solutions (Fig. 6, lines
4–8). The fluorescence band in the spectrum recorded
in nonpolar 1,4-dioxane exhibits the highest intensity,
which indicates the highest quantum yield of fluores-
cence.
Fig. 5. Absorption temperature spectra of VI (1.07 × 10−5 M,
10 mM Tris-HCl buffer) registered in the range of 25–
90◦C; 25◦C (1), 35◦C (2), 45◦C (3), 55◦C (4), 65◦C (5),
75◦C (6), 85◦C (7), 90◦C (8); (a) whole spectrum, (b)
relevant segment of spectrum.

1236
A. Gꢀluszyn´ska et al./Chemical Papers 67 (9) 1231–1239 (2013)
Fig. 6. Emission spectra of VI (1.36 × 10−6 M) in selected or-
ganic solvents and aqueous solutions at ambient tem-
perature: 1,4-dioxane (1), CHCl3 (2), CH2Cl2 (3),
EtOH (4), DMSO (5), CH3CN (6), Tris-HCl buffer (7),
H2O (8), toluene (9).
Fig. 7. Emission spectra of VI (1.36 × 10−6 M) in CHCl3 (1),
Fig. 8. Absorption spectra of VI. Conditions: [VI]
=
1.53 × 10−5 M, 10 mM Tris-HCl buffer; (a) λirr = 451
nm, exposure time: 0 h (1), 1 h (2); (b) λirr = 272 nm,
exposure time: 0 h (2), 1 h (3).
10 vol. % of toluene in CHCl3 (2) and toluene (3).
The use of another nonpolar solvent, toluene, re-
sulted in the fluorescence quenching as the ligand
molecules undergo strong association in this solvent
(UV-VIS spectrum) and although they absorb energy
they are not able to emit. Aggregation significantly de-
creased the fluorescence intensity and the obtained re-
sults confirmed the changes in the absorption spectra.
As expected, the emission spectrum of the ligand in
CHCl3 with a small addition of toluene (10 vol. %) is
less intense (19 %) than the ligand spectrum recorded
in pure CHCl3 (∆λ = 3 nm) (Fig. 7).
One of the features of VI is the presence of a double
bond which offers a possibility of the ligand’s structure
control by light (cis/trans photoisomerisation). Our
preliminary results support this idea. Upon a one hour
irradiation performed with the light of λirr = 451 nm,
the absorption spectrum of the ligand changed. The
long-wavelength maximum of absorption at 480 nm
decreased with no hypsochromic shift of the band with
a simultaneous increase of the short-wavelength band
at 292 nm and the isosbestic point at 359 nm providing
a two-component system (Fig. 8a). These changes sug-
gest the ligand transformation from trans to cis; the
latter form is characterised by a much lower molar ab-
sorption coefficient. During irradiation of the sample
with the light of λirr = 272 nm, opposite changes were
observed: the long-wavelength maximum of absorption
at 480 nm increased with a simultaneous decrease of
the short-wavelength band at 292 nm. These changes
suggest the return of the cis form of VI to the trans
form (Fig. 8b). Further studies are in progress.
Preliminary studies on the DNA binding affin-
ity of VI were performed by the spectrophotomet-
ric titration method on calf thymus DNA as a
double-stranded DNA (dsDNA) and 22-mer oligonu-
cleotide (5ꢀ-AGGGTTAGGGTTAGGGTTAGG G-3ꢀ)

A. Gꢀluszyn´ska et al./Chemical Papers 67 (9) 1231–1239 (2013)
1237
Fig. 9. Spectrophotometric titration of VI with ctDNA. Con-
ditions: [dye] = 10.9 µM, Tris-HCl buffer (10 mM, pH
7.2), 100 mM NaCl, [oligonucleotide] = 0–480 µM, Tris-
HCl buffer (10 mM, pH 7.2), 100 mM NaCl.
with a sequence related to that of human telom-
ere DNA (G4 DNA), which can form intramolecu-
lar G-quadruplex structures by folding of a single
oligonucleotide molecule. In these experiments, dis-
tinct changes in the absorption spectra of the lig-
and were observed (bathochromic shift, hypochromic-
ity, isosbestic point). The interaction with ctDNA re-
sulted in substantial hypochromicity (ca. 32 %) and a
rather considerable red shift of the absorption max-
imum (∆λmax = 19 nm). The isosbestic point was
clearly recognised at 472 nm (Fig. 9). Such behaviour
is a commonly observed in dyes undergoing interca-
lation (Wilson et al., 1989; Czarny et al., 1995) thus
suggesting an intercalation binding mode also in case
of VI. Spectral changes upon the titration with G4
DNA in the presence of cations Na+ and K+, which
stabilise the structure of G-quadruplexes, were similar
to those shown above, as it can be seen in Figs. 10a
and 10b. The hypochromicity was comparable for Na+
and more pronounced for K+ (34 % and 56 %, re-
spectively) while a clear shift toward the longer wave-
lengths (∆λmax = 10 nm for Na+ and 32 nm for K+)
was observed. The isosbestic point was observed only
for the titration in the presence of Na+ cations at
495 nm.
Such spectral changes are typical of intercalators,
which leads to a conclusion that VI is associated
with ctDNA by intercalation while in 22Htel, the end-
stacking type interactions take place. Titration data
were analysed in order to calculate DNA-binding pa-
rameters. The results obtained were analysed accord-
ing to the simple Scatchard method. Analysis of the
data did not permit unambiguous determination of
the number of binding sites, complex stability con-
stant (K) and the number of ligand molecules per nu-
cleotide (n). According to literature, determination of
these parameters is not always possible. However, sig-
nificant spectral changes clearly indicated a high affin-
Fig. 10. Spectrophotometric titration of VI with G4 DNA.
Conditions: (a) [dye] = 8.03 µM, Tris-HCl buffer
(10 mM, pH 7.2), 100 mM NaCl, [oligonucleotide] =
0–5 µM, Tris-HCl buffer (10 mM, pH 7.2), 100 mM
NaCl; (b) [dye] = 8.03 µM, Tris-HCl buffer (10 mM,
pH 7.2), 150 mM KCl, [oligonucleotide] = 0–15 µM,
Tris-HCl buffer (10 mM, pH 7.2), 150 mM KCl.
ity of the BMVC ligand to the sequence related to
that of human telomere DNA (Hum24) (Chang et al.,
2003b, 2004a, 2006a). Non-linearity in the relationship
of free and bound ligands proves the complexity of the
process in which both nonspecific and specific binding
sites are involved. Further studies on this subject are
in progress.
Investigation of the processes of interaction of new
ligands in the presence of double-, triple- and tetra-
stranded DNA, and the determination of mechanisms
of the above processes should provide interesting ex-
perimental data for the design of effective DNA lig-
ands potentially active in anticancer therapy and use-
ful as fluorescence probes for structural studies of
DNA or detection of trace amounts of DNA.

1238
A. Gꢀluszyn´ska et al./Chemical Papers 67 (9) 1231–1239 (2013)
Acknowledgements. This research project is realised within
Dias, N., Jacquemard, U., Baldeyrou, B., Tardy, C., Lansi-
aux, A., Colson, P., Tanious, F., Wilson, W. D., Routier,
S., Mérour, J. Y., & Bailly, C. (2004). Targeting DNA with
novel diphenylcarbazoles. Biochemistry, 43, 15169–15178.
DOI: 10.1021/bi048474o.
the PARENT-BRIDGE programme of the Foundation for Pol-
ish Science, co-financed by the European Union, Regional De-
velopment Fund.
Dumat, B., Bordeau, G., Faurel-Paul, E., Mahuteau-Betzer, F.,
Saettel, N., Bombled, M., Metgé, G., Charra, F., Fiorini-
Debuisschert, C., & Teulade-Fichou, M. P. (2011). N-phenyl-
carbazole-based two-photon fluorescent probes: Strong se-
quence dependence of the duplex vs quadruplex selectiv-
ity. Biochimie, 93, 1209–1218. DOI: 10.1016/j.biochi.2011.05.
035.
Folini, M., Gandellini, P., & Zaffaroni, N. (2009). Targeting the
telosome: Therapeutic implications. Biochimica et Biophys-
ica Acta, 1792, 309–316. DOI: 10.1016/j.bbadis.2009.01.014.
Franceschin, M. (2009). G-quadruplex DNA structures and
organic chemistry: More than one connection. European
Journal of Organic Chemistry, 2009, 2225–2238. DOI:
10.1002/ejoc.200801196.
References
Brooks, T, A., & Hurley, L. H. (2010). Targeting MYC expres-
sion through G-quadruplexes. Genes & Cancer, 1, 641–649.
DOI: 10.1177/1947601910377493.
Chang, C. C., Wu, J. Y., & Chang, T. C. (2003a). A carbazole
derivative synthesis for stabilizing the quadruplex structure.
Journal of the Chinese Chemical Society, 50, 185–188.
Chang, C. C., Wu, J. Y., Chien, C. W., Wu, W. S., Liu, H.,
Kang, C. C., Yu, L. J., & Chang, T. C. (2003b). A flu-
orescent carbazole derivative: High sensitivity for quadru-
plex DNA. Analytical Chemistry, 75, 6177–6183. DOI:
10.1021/ac034789i.
Chang, C. C., Kuo, I. C., Lin, J. J., Lu, Y. C., Chen, C. T., Back,
H. T., Lou, P. J., & Chang, T. C. (2004a). A novel carbazole
derivative, BMVC: a potential antitumor agent and fluores-
cence marker of cancer cells. Chemistry & Biodiversity, 1,
1377–1384. DOI: 10.1002/cbdv.200490100.
Gellert, M., Lipsett, M. N., & Davies, D. R. (1962). Helix forma-
tion by guanylic acid. Proceedings of the National Academy
of Sciences of the United States of America, 48, 2013–2018.
Glꢀuszyn´ska, A., Bajor, K., Czerwin´ska, I., Kalet, D.,
&
Chang, C. C., Kuo, I. C., Ling, I. F., Chen, H. T., Chen, H.
C., Lou, P. J., Lin, J. J., & Chang, T. C. (2004b). Detec-
tion of quadruplex DNA structures in human telomeres by
a fluorescent carbazole derivative. Analytical Chemistry, 76,
4490–4494. DOI: 10.1021/ac049510s.
Chang, C. C., Chu J. F., Kao, F. J., Chiu, Y. C., Lou, P. J.,
Chen, H. C., & Chang, T. C. (2006a). Verification of an-
tiparallel G-quadruplex structure in human telomeres by us-
ing two-photon excitation fluorescence lifetime imaging mi-
croscopy of the 3,6-bis(1-methyl-4-vinylpyridinium)carbazole
diiodide molecule. Analytical Chemistry, 78, 2810–2815.
DOI: 10.1021/ac052218f.
Chang, C. C., Chu, J. F., Kuo H. H., Kang, C. C., Lin, S. H., &
Chang, T. C. (2006b). Solvent effect on photophysical prop-
erties of a fluorescence probe: BMVC. Journal of Lumines-
cence, 119–120, 84–90. DOI: 10.1016/j.jlumin.2005.12.016.
Chang, C. C., Chien, C. W., Lin, Y. H., Kang, C. C., &
Chang, T. C. (2007). Investigation of spectral conversion
of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titra-
tion by a G-quadruplex recognizer BMVC molecule. Nucleic
Acids Research, 35, 2846–2860. DOI: 10.1093/nar/gkm155.
Juskowiak, B. (2010). The synthesis and spectral properties
of new DNA binding ligands. Tetrahedron Letters, 51, 5415–
5418. DOI: 10.1016/j.tetlet.2010.07.180.
Glꢀuszyn´ska, A., Rajczak, E., & Juskowiak, B. (2012). The syn-
thesis and spectral properties of new carbazole ligand, po-
tential DNA intercalator. In J. Markoš (Ed.), Proceedings of
the 39th International Conference of the Slovak Society of
Chemical Engineering, May 21–25, 2012 (P. 098, pp 1349–
1354). Tatranské Matliare, Slovakia: Slovak Society of Chem-
ical Engineering.
Haugland, R. P. (1996). Handbook of fluorescent probes and
research chemicals (6th ed.). Eugene, OR, USA: Molecular
Probes, Inc.
Hotzel, C., Marotto, A., & Pindur, U. (2002). Design, synthesis,
DNA-binding and cytotoxicity evaluation of new potential
combilexines. European Journal of Medicinal Chemistry, 37,
367–378. DOI: 10.1016/s0223-5234(02)01349-1.
Huang, F. C., Chang, C. C., Lou, P. J., Kuo, I. C., Chien,
C. W., Chen, C. T., Shieh, F. Y., Chang, T. C., & Lin,
J. J. (2008). G-quadruplex stabilizer 3,6-bis(1-methyl-4-
vinylpyridinium)carbazole diiodide induces accelerated sene-
scence and inhibits tumorigenic properties in cancer cells.
Molecular Cancer Research, 6, 955–964. DOI: 10.1158/1541-
7786.MCR-07-0260.
Jain, A. K., & Bhattacharya, S. (2011). Interaction of G-
quadruplexes with nonintercalating duplex-DNA minor
groove binding ligands. Bioconjugate Chemistry, 22, 2355–
2368. DOI: 10.1021/bc200268a.
Juskowiak, B., Ohba, M., Sato, M., Takenaka, S., Takagi, M., &
Kondo, H. (1999). Photoisomerizable DNA ligands. Spectral
and electrochemical properties and base-pair selectivity of
binding of bis[2-(1-alkylpyridinium-4-yl)vinyl]benzene dyes.
Bulletin of the Chemical Society of Japan, 72, 265–277. DOI:
10.1246/bcsj.72.265.
Juskowiak, B., Chudak, M., Takagi, N., & Takagi, M. (2002).
Photoisomerizable DNA ligands. Photoizomerization of an-
thrylvinylpyridinium derivatives. Polish Journal of Chem-
istry, 76, 83–93.
Chang, T. C.,
& Chang, C. C. (2010). Detection of G-
quadruplexes in cells and investigation of G-quadruplex
structure of d(T2AG3)4 in K+ solution by a carbazole deriva-
tive: BMVC. In P. Baumann (Ed.), G-quadruplex DNA:
Methods and protocols (Series: Methods in molecular biol-
ogy, Vol. 608, pp. 183–206). New York, NY, USA: Humana
Press. DOI: 10.1007/978-1-59745-363-9 12.
Cuesta, J., Read, M. A., & Neidle, S. (2003). The design of G-
quadruplex ligands as telomerase inhibitors. Mini-Reviews in
Medicinal Chemistry, 3, 11–21.
Czarny, A., Boykin, D. W., Wood, A. A., Nunn, C. M., Nei-
dle, S., Zhao, M., & Wilson, W. D. (1995). Analysis of van
der Waals and electrostatic contributions in the interactions
of minor groove binding benzimidazoles with DNA. Jour-
nal of the American Chemical Society, 117, 4716–4717. DOI:
10.1021/ja00121a034.
Davis, J. T. (2004). G-quartets 40 years later: From 5ꢀ-GMP
to molecular biology and supramolecular chemistry. Ange-
wandte Chemie International Edition, 43, 668–698. DOI:
10.1002/anie.200300589.
Kelland, L. R. (2005). Overcoming the immortality of tumour
cells by telomere and telomerase based cancer therapeutics
– current status and future prospects. European Journal of
Cancer, 41, 971–979. DOI: 10.1016/j.ejca.2004.11.024.
Kerwin, S. M. (2000). G-quadruplex DNA as a target for drug
design. Current Pharmaceutical Design, 6, 441–471.
De Cian, A., Lacroix, L., Douarre, C., Temime-Smaali, N.,
Trentesaux, C., Riou, J. F., & Mergny, J. L. (2008). Target-
ing telmeres and telomerase. Biochimie, 90, 131–155. DOI:
10.1016/j.biochi.2007.07.011.

A. Gꢀluszyn´ska et al./Chemical Papers 67 (9) 1231–1239 (2013)
1239
Kim, H. K., Ryu, M. K., Kim, K. D., Lee, S. M., Cho, S.
W., & Park, J. W. (1998). Tunable electroluminescence from
silicon-containing poly(p-phenylenevinylene)-related copoly-
mers with well-defined structures. Macromolecules, 31, 1114–
1123. DOI: 10.1021/ma9711553.
Tanious, F. A., Ding, D., Patrick, D. A., Bailly, C., Tid-
well, R. R., & Wilson, W. D. (2000). Effects of compound
structure on carbazole dication–DNA complexes: Tests of
the minor-groove complex models. Biochemistry, 39, 12091–
12101. DOI: 10.1021/bi001236i.
Li, J., Li, D., Han, Y., Shuang, S.,
&
Dong, C. (2009).
Tanious, F. A., Wilson, W. D., Patrick, D. A., Tidwell, R. R.,
Colson, P., Houssier, C., Tardy, C., & Bailly, C. (2001).
Sequence-dependent binding of bis-amidine carbazole dica-
tions to DNA. European Journal of Biochemistry, 268, 3455–
3464. DOI: 10.1046/j.1432-1327.2001.02242.x.
Synthesis of 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-
2-pyrazoline and its use as DNA probe. Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy, 73, 221–
225. DOI: 10.1016/j.saa.2009.01.019.
Ou, T. M., Lu, Y. J., Tan, J. H., Huang, Z. S., Wong, K.
Y., & Gu, L. Q. (2008). G-quadruplexes: Targets in an-
ticancer drug design. ChemMedChem, 3, 690–713. DOI:
10.1002/cmdc.200700300.
Patrick, D. A., Boykin, D. W., Wilson, W. D., Tanious, F. A.,
Spychala, J., Bender, B. C., Hall, J. E., Dykstra, C. C., Ohe-
meng, K. A., & Tidwell, R. R. (1997). Anti-Pneumocystis
carinii pneumonia activity of dicationic carbazoles. Euro-
pean Journal of Medicinal Chemistry, 32, 781–793. DOI:
10.1016/s0223-5234(99)80064-6.
Tsai, Y. L., Chang, C. C., Kang, C. C., & Chang, T. C. (2007).
Effect of different electronic properties on 9-aryl-substituted
BMVC derivatives for new fluorescence probes. Journal of
Luminescence, 127, 41–47. DOI: 10.1016/j.jlumin.2007.02.
054.
White, E. W., Tanious, F., Ismail, M. A., Reszka, A. P., Neidle,
S., Boykin, D. W., & Wilson, W. D. (2007). Structure-specific
recognition of quadruplex DNA by organic cations: Influence
of shape, substituents and charge. Biophysical Chemistry,
126, 140–153. DOI: 10.1016/j.bpc.2006.06.006.
Qu, J., Morita, R., Ashitaka, H., Ogata, N.,
&
Masuda,
Wilson, W. D., Tanious, F. A., Barton, H. J., Jones, R.,
Strekowski, L., & Boykin, D. W. (1989). Binding of 4ꢀ,6-
diamidino-2-phenylindole (DAPI) to GC and mixed se-
quences in DNA: Intercalation of a classical groove-binding
molecule. Journal of the American Chemical Society, 111,
5008–5010. DOI: 10.1021/ja00195a080.
Xu, T. H., Lu, R., Qiu, X. P., Liu, X. L., Xue, P. C., Tan, C. H.,
Bao, C. Y., & Zhao, Y. Y. (2006). Synthesis and character-
ization of carbazole-based dendrimers with porphyrin cores.
European Journal of Organic Chemistry, 2006, 4014–4020.
DOI: 10.1002/ejoc.200600356.
Yang, D. Y., Chang, T. C., & Sheu, S. Y. (2007). Interaction be-
tween human telomere and a carbazole derivative: A molecu-
lar dynamics simulation of a quadruplex stabilizer and telom-
erase inhibitor. The Journal of Physical Chemistry A, 111,
9224–9232. DOI: 10.1021/jp071963o.
Zhang, F. F., Gan, L. L., & Zhou, C. H. (2010). Synthesis, an-
tibacterial and antifungal activities of some carbazole deriva-
tives. Bioorganic & Medicinal Chemistry Letters, 20, 1881–
1884. DOI: 10.1016/j.bmcl.2010.01.159.
T. (2008). DNA–lipid complexes carrying carbazole and
triphenylamine moieties: Synthesis, and chiroptical and
photoelectronic properties. Polymer, 49, 3663–3670. DOI:
10.1016/j.polymer.2008.06.039.
Riou, J. F. (2004). G-quadruplex interacting agents target-
ing the telomeric G-overhang are more than simple telom-
erase inhibitors. Current Medicinal Chemistry - Anti-Cancer
Agents, 4, 439–443. DOI: 10.2174/1568011043352740.
Ryu, H., Subramanian, L. R., & Hanack, M. (2006). Photo-
and electroluminescent properties of cyano-substituted styryl
derivatives and synthesis of CN–PPV model compounds con-
taining an alkoxy spacer for OLEDs. Tetrahedron, 62, 6236–
6247. DOI: 10.1016/j.tet.2006.04.051.
Saengkhae, C., Salerno, M., Ad`es, D., Siove, A., Le Moyec, L.,
Migonney, V., & Garnier-Suillerot, A. (2007). Ability of car-
bazole salts, inhibitors of Alzheimer β-amyloid fibril forma-
tion, to cross cellular membranes. European Journal of Phar-
macology, 559, 124–131. DOI: 10.1016/j.ejphar.2007.01.005.
Song, Y., Di, C. A., Wei, Z., Zhao, T., Xu, W., Liu, Y., Zhang,
D., & Zhu, D. (2008). Synthesis, characterization, and field-
effect transistor properties of carbazolenevinylene oligomers:
From linear to cyclic architectures. Chemistry - A European
Journal, 14, 4731–4740. DOI: 10.1002/chem.200800008.
Spychalꢀa, J. (2009). Selective cytostatic and cytotoxic anti-
cancer effects of bisfunctional agents: A strategy for the de-
sign of DNA binding agents. Cancer Letters, 281, 203–212.
DOI: 10.1016/j.canlet.2009.02.026.
Zhang, Y., Wang, L., Wada, T., & Sasabe, H. (1996). Syn-
thesis and characterization of novel hyperbranched poly-
mer with dipole carbazole moieties for multifunctional
materials. Journal of Polymer Science: Part A: Poly-
mer Chemistry, 34, 1359–1363. DOI: 10.1002/(SICI)1099-
0518(199605)34:7<1359::AID-POLA26>3.0.CO;2-7.
Sun, D., Thompson, B., Cathers, B. E., Salazar, M., Kerwin, S.
M., Trent, J. O., Jenkins, T. C., Neidle, S., & Hurley, L. H.
(1997). Inhibition of human telomerase by a G-quadruplex-
interactive compound. Journal of Medicinal Chemistry, 40,
2113–2116. DOI: 10.1021/jm970199z.