Dimeric 1,3-Phenylene-bis(piperazinyl benzimidazole)s: Synthesis and structure-activity investigations on their binding with human telomeric G-quadruplex DNA and telomerase inhibition properties

Article abstract of DOI:10.1021/jm200860b

Ligand-induced stabilization of G-quadruplex structures formed by the human telomeric DNA is an active area of research. The compounds which stabilize the G-quadruplexes often lead to telomerase inhibition. Herein we present the results of interaction of new monomeric and dimeric ligands having 1,3-phenylene-bis(piperazinyl benzimidazole) unit with G-quadruplex DNA (G4DNA) formed by human telomeric repeat d[(G₃T₂A) ₃G₃]. These ligands efficiently stabilize the preformed G4DNA in the presence of 100 mM monovalent alkali metal ions. Also, the G4DNA formed in the presence of low concentrations of ligands in 100 mM K⁺ adopts a highly stable parallel-stranded conformation. The G-quadruplexes formed in the presence of the dimeric compound are more stable than that induced by the corresponding monomeric counterpart. The dimeric ligands having oligo-oxyethylene spacers provide much higher stability to the preformed G4DNA and also exert significantly higher telomerase inhibition activity. Computational aspects have also been discussed.

Full text of DOI:10.1021/jm200860b

Article
pubs.acs.org/jmc
Dimeric 1,3-Phenylene-bis(piperazinyl benzimidazole)s: Synthesis
and Structure−Activity Investigations on their Binding with Human
Telomeric G-Quadruplex DNA and Telomerase Inhibition Properties
Akash K Jain,† Ananya Paul,† Basudeb Maji,† K. Muniyappa,§ and Santanu Bhattacharya*,†,‡
†Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India
‡Chemical Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560012, India
§Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
S
* Supporting Information
ABSTRACT: Ligand-induced stabilization of G-quadruplex
structures formed by the human telomeric DNA is an active
area of research. The compounds which stabilize the G-
quadruplexes often lead to telomerase inhibition. Herein we
present the results of interaction of new monomeric and
dimeric ligands having 1,3-phenylene-bis(piperazinyl benzimi-
dazole) unit with G-quadruplex DNA (G4DNA) formed by
human telomeric repeat d[(G3T2A)3G3]. These ligands
efficiently stabilize the preformed G4DNA in the presence of
100 mM monovalent alkali metal ions. Also, the G4DNA
formed in the presence of low concentrations of ligands in 100
mM K+ adopts a highly stable parallel-stranded conformation. The G-quadruplexes formed in the presence of the dimeric
compound are more stable than that induced by the corresponding monomeric counterpart. The dimeric ligands having oligo-
oxyethylene spacers provide much higher stability to the preformed G4DNA and also exert significantly higher telomerase
inhibition activity. Computational aspects have also been discussed.
stranded.11 However, there has been a considerable debate
INTRODUCTION
A characteristic feature of the most biological polymers is their
■
regarding the exact structure of such DNA in a physiologically
relevant K+-rich environment. For example, the crystal structure
ability to fold into specific three-dimensional structures under
of G4DNA in the presence of K+ has been shown to be
physiological ionic conditions, and these structures are
propeller-like and parallel-stranded.12 However, this structure
functionally definitive. The telomeric DNA consists of simple
differs from the structure adopted under the physiological
tandem repeats of guanine (G)-rich sequences, which are
conditions. The favored conformation under the physiological
typified by the hexanucleotide d(5′-TTAGGG)n in verte-
brates.1,2 In eukaryotes, the telomere length of the repeat
environment was reported to be a hybrid structure having
mixed parallel/antiparallel strands and three G-tetrads within
TTAGGG is in the range of 5−15 kb. In this region of
telomeric DNA, the tandem repeats of the G-rich sequences
the G4DNA.13 Thus it was suggested that the crystal structure
associated with the parallel-stranded conformation may not be
fold into the G-quadruplex structures. All G4DNA structures
relevant in the biologically active form.14 Recently, a stable,
contain a basic repeating and stacking motif, the G-quartet
basket-type, antiparallel stranded G4DNA structure having only
structure, which is formed by the four guanine bases and is held
two G-tetrad layers has been discovered in K+ solution.13d,15
The human telomeric DNA sequence adopts parallel-stranded
conformation in K+ solution in the presence of high
concentrations of PEG.16 This effect could be due to the
dehydration of Hoogsteen hydrogen bonds caused by
molecular crowding.17 The conformation of the G-quadruplex
DNA may be altered by an interaction with the G-quadruplex-
specific ligands.18 Most of the G-quadruplex DNA-specific
compounds indeed have a central planar pharmacophore
capable of binding to the guanine tetrads via π−π stacking
in a plane by Hoogsteen type hydrogen bonds.3−6 Telomerase,
a ribonucleoprotein complex of about 170 kDa, is up-regulated
in about 80−90% of human tumors and is undetectable in most
of the normal somatic cells.7 The inhibition of telomerase
activity overcomes the ability of cells to proliferate and leads to
cell death. Telomerase enzyme is thus a highly attractive target
for the selective anticancer therapy by the G-quadruplex-
specific ligands.4,8 In vitro experiments have shown that if the
telomeric DNA is folded into G-quadruplex structures, it
becomes insensitive to the elongation by telomerase.8,9
The intracellular concentrations of K+ and Na+ in animal
cells are up to 150 and 15 mM, respectively.10 Structure of the
human telomeric G4DNA in Na+ solution is antiparallel-
Received: July 1, 2011
Published: March 27, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of the compounds used in this study.
a
Scheme 1
a
(a) p-TsCl, NaOH, THF/H2O; (b) K2CO3, CH3CN, reflux; (c) LAH, THF, rt; (d) PCC, DCM or DCM/THF, rt.
interactions. These also have side chains that can offer different
modes of hydrogen bonding interactions directed toward the
G-quadruplex loops and grooves.19
the dimeric motif to act as a potent G4DNA stabilizer. Indeed
the dimeric forms of DNA targeting ligands are worthwhile for
their design and synthesis. However, only a couple of studies
are known where such ligands have been evaluated toward their
binding of G4DNA. Recently, some dimeric G-quadruplex
ligands, having higher quadruplex affinity than the correspond-
ing monomeric ligands, have been developed. BRACO19 based
dimers were found to have lower IC50 values for telomerase
inhibition,24 and dimeric ligands having macrocyclic hexa-
oxazole units were found to have lower IC50 values for G-
quadruplex stabilization as determined by PCR stop assay.25
However, these studies did not examine the preferred structure
Bisbenzimidazole based Hoechst 33258 binds to the G4DNA
assembled from the promoter region of human c-myc,20
telomeric G-quadruplex21 and some of its analogues have
been found to stabilize triple-helical DNA at acidic pH.22
Previously, we have reported the ability of small synthetic
molecules based on benzimidazoles, some of which bind duplex
DNA efficiently and few others inhibit the topoisomerase
activity.23 In this paper, we report the synthesis of a series of
monomeric and dimeric derivatives to elucidate the minimum
structural prerequisites and the ability of a molecule containing
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of the G4DNA−ligand complex under the physiological
environment.
Scheme 2
We report herein the G4DNA formation by human telomere
sequence d[G3(TTAG3)3] (abbreviated as Hum21), its
stabilization and structural alteration by six ligands having
1,3-phenylene-bis (piperazinyl benzimidazole) unit (mono-
meric ligands 1a and 1b and dimeric ligands 2a−2d) (Figure
1). The central unit of these molecules is “V”-shaped, which has
been shown to possess higher selectivity for the G4DNA than
the corresponding linear isomeric ligands.26 Monomeric ligands
1a and 1b have already been shown to stabilize and alter the
topology of the G4DNA formed by the DNA sequence
d(T2G4)4, a telomeric repeat from Tetrahymena thermophilia.27
Importantly, these compounds have >100 times greater affinity
for the G4DNA than the AT-rich duplex DNA. Furthermore,
these ligands induce G4DNA formation in the absence of any
added cations.27 The intrinsic fluorophoric properties of these
ligands make the evaluation of their G-quadruplex binding
properties particularly convenient. Therefore, we have
developed the corresponding dimeric compounds and inves-
tigated in detail their abilities to stabilize G4DNA (Hum21)
using a number of different ways. We present the results of
these findings in this paper.
mixed parallel/antiparallel strands within the G4DNA.13 We
used three buffer systems, one having LiCl (10 mM sodium
cacodylate having 100 mM LiCl and 0.1 mM EDTA, pH 7.4)
and other two were made of either NaCl or KCl salts (10 mM
Tris-HCl having 100 mM of either NaCl or KCl and 0.1 mM
EDTA, pH 7.4). We used heat annealing conditions to form the
G4DNA of the sequence d[G3(T2AG3)3] (abbreviated as
Hum21, Materials and Methods). CD spectra obtained from the
G4DNA formed by Hum21 in NaCl and KCl are similar to the
reported ones (Figure 2).16 G4DNA formed in KCl solution
RESULTS AND DISCUSSION
■
Synthesis. The following considerations were made while
designing suitable ligands for achieving high stabilization of the
G4DNA. Bisbenzimidazole compounds are known to be
permeable across cell and nuclear membranes and have been
used widely in cytometry and chromosomal staining.28
Compounds having “V”-shaped central planar core have high
affinity toward the G4DNA.26,27,29 Polyethylene glycol based
polymers, e.g., have both hydrophilic as well as lipophilic
properties. Hence incorporation of oligo-oxyethylene based
linker units between two bisbenzimidazole pharmacophoric
units would further increase its cell permeability and make it
more biocompatible. PEG also has other biological relevance
along with its anticancer properties.30 Consequently, we have
synthesized first a member of some monomeric ligand
molecules having central “V”-shaped planar core and the
corresponding dimers having variable lengths of oligo-oxy-
ethylene linkers connecting each monomeric units.
Briefly, for the synthesis of dimers, the glycols 3 or 4 were
converted into respective ditosylates, which were then reacted
with 5-ethoxy-isophthalic acid dimethyl ester to give the
respective tetraesters 7 and 8 in 85% and 86% yields,
respectively. Each compound 7 and 8 was then separately
oxidized using PCC to obtain the key tetraaldehydes 11 and 12
in 70% and 72% yields, respectively (Scheme 1). Tetraalde-
hydes 11 and 12 were then subjected to oxidative coupling with
31
diamines 13 or 1427 in the presence of Na2S2O5 to give the
final dimeric bisbenzimidazoles (2a−2d) in 60−62% yields
(Scheme 2). Corresponding monomeric ligands 1a and 1b
were synthesized as described earlier.27
Figure 2. CD spectra due to G4DNA formed by human telomeric
sequence Hum21 (4 μM strand concentration) in the presence of
indicated ions (100 mM). This contains 10 mM sodium cacodylate
a
G-Quadruplex Formation. Monovalent ions, e.g., Na+ and
K+, are known to induce and stabilize the G4DNA structures.
Li+ ions on the other hand induce the G4DNA formation, but
they have very little effect on the G4DNA stability.32 In Na+
solution, the sequence d[AG3(T2AG3)3] folds intramolecularly
into a G-tetraplex DNA stabilized by three stacked G-tetrads
which are connected by two lateral loops and a central diagonal
loop, leading to an antiparallel geometry.11 The favored
conformation under a physiological K+ environment has
buffer and 100 mM LiCl.
has one small negative peak near 240 nm, one positive peak
near 292 nm, and shoulders near 250 and 270 nm, similar to
those reported for the human telomeric DNA in K+ solution.16
For the CD spectra of the G4DNA formed in LiCl buffer, the
positions of the spectral peaks matched with those of the Na+
quad but for the former the positive peaks at 295 and 245 nm
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Figure 3. Relative increments in the fluorescence emission intensity due to the indicated ligands (400 nM of monomers or 200 nM of dimers) in
LiCl buffer (10 mM sodium cacodylate having 100 mM LiCl and 0.1 mM EDTA, pH 7.4) (A) and in KCl buffer (10 mM Tris-HCl having 100 mM
KCl and 0.1 mM EDTA, pH 7.4) (B). G4DNA formed by Hum21 was added from the stock of 4 μM strand concentration to the solution of the
individual ligand in the cuvette.
were more intense while the negative peak at 264 nm was less
intense than that of the latter (Figure 2).
intervening oligo-oxyethylene linker chain adopts a crown ether
type of structure in presence of these alkali metal ions in their
energy minimized states (Figure S7, Supporting Information).
How does the formation of a supramolecular complex with a
metal ion influence their binding with the G4DNA? To answer
this, we also carried out a control experiment in absence of
preformed G4DNA at the identical salt concentration. In these
circumstances, the ligands showed comparable fluorescence
quenching in presence of Li+ and Na+ ions (Figure S9,
Supporting Information). Ligands 2c and 2d did not, however,
form supramolecular structures with K+ ions. Under these
circumstances, they showed efficient binding with the G4DNA
in K+ solution. However, a recent study shows that the
formation of a ligand−metal supramolecular complex should
not alter the activity of the ligand with the G4DNA or the
duplex DNA.33
Next, we have determined the binding constants of the
ligands with the preformed Hum21 G4DNA in KCl solution
using the method described earlier.26,27 Monomeric ligand 1b
provided higher stabilization of the G4DNA than 1a,
presumably because of the presence of an electron-donating
ethoxy group (Table 1).26 Among dimeric ligands, 2c and 2d,
the one having longer tetraethylene glycol linker provided
higher stabilization of the G4DNA than 2a and 2b having
shorter linkers. Dimeric ligand 2d exerted the highest
stabilization (Table 1).
Fluorescence and UV−Vis Titrations. Each of the
monomeric and dimeric ligands displayed low fluorescence
emission in Tris-HCl buffer having 100 mM LiCl (that also has
10 mM Na+) at pH 7.4. During the fluorescence spectral
titrations involving DNA, addition of preformed Hum21
G4DNA to either 1a or 1b induced significant enhancements
in the fluorescence emission intensity, with the saturation point
near [DNA]:[ligand] ratio = 0.1 or [ligand]:[DNA] ratio = 10
(Figure 3A). In contrast, the dimeric compounds showed
considerably less increments in the fluorescence intensity upon
addition of preformed Hum21 G4DNA under the same
conditions. While only 2a showed a moderate increment,
ligands 2b and 2c showed much less increments in fluorescence
than 2a. In the case of 2d, the fluorescence intensity first
decreased and then increased slightly during titration with the
above G4DNA (Figure 3A). These findings suggest that in LiCl
solution, the monomeric ligands 1a and 1b bind with the
preformed Hum21 G4DNA and stabilize the latter more
effectively than their dimeric counterparts.
However, in 100 mM KCl solution, the dimeric ligands 2c
and 2d showed significant fluorescence enhancements (Figure
3B). Saturation in the fluorescence emission occurred at
[DNA]:[ligand] ratio = 0.16. In the case of the dimeric ligands,
the two monomeric fluorescent ligand units are connected by
an oligo-oxyethelene linker, which is capable to adopt a “crown
ether” like conformation in the presence of alkali metal ion and
transforms its conformation from a linear to a “hairpin”-like
motif. Dimeric ligands 2a and 2b have a triethylene glycol
based linker which is capable of forming a complex with the Li+
ion and adopt a 12-crown-4 like structure (Figure S8,
Supporting Information). Using fluorescence titrations, the
association constant of binding of 2b with Li+ ion was found to
Table 1. Dissociation Constant (KD) of All Four Compounds
a
with Preformed Hum21 G4DNA Formed in KCl Buffer
KD (107 M−1
)
ligand
Hum21 G4DNA
1a
1b
2a
2b
2c
2d
4.25 0.1
8.49 0.2
6.04 0.2
6.54 0.6
9.15 0.6
12.28 0.3
be 5.5
0.5 × 105 M−1 (method described in Supporting
Information). The other dimeric ligands, 2c and 2d, possess a
longer tetra-ethylene glycol based linker which is capable of
forming a complex with Na+ ion to adopt a 15-crown-5 like
shape (Figure S8, Supporting Information). The association
constant of binding of 2d with Na+ ion was found to be 2.3
0.1 × 105 M−1. Indeed, a computational study of 2b and 2d in
presence of Li+ and Na+ ions respectively also suggests that the
a
Binding assays were performed with preformed Hum21 G4DNA in 10
mM Tris-HCl, having 100 mM KCl and 0.1 mM EDTA buffer, at pH
7.3.
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Figure 4. (A) CD titrations of the preformed Hum21 G4DNA (4 μM strand concentration) with increasing amount of 2d (0, 20, 40, 60, 80, and 100
μM), respectively, in KCl buffer (10 mM Tris-HCl having 100 mM KCl and 0.1 mM EDTA, pH 7.4). (B) CD spectral profiles of the Hum21 (4 μM
strand concentration) G4DNA formed in presence of 0, 20, and 40 μM of 2d (heated at 90 °C for 5 min and then cooled slowly) in 100 mM KCl
buffer.
Figure 5. (A) CD titrations of the preformed Hum21 G4DNA (4 μM strand concentration) with increasing amount of 1b (0, 40, 60, and 100 μM,
respectively) in KCl buffer (10 mM Tris-HCl having 100 mM KCl and 0.1 mM EDTA, pH 7.4). (B) CD spectral profiles of the Hum21 (2 μM
strand concentration) G4DNA formed in presence of 0 and 40 μM of 1b (first heated at 90 °C for 5 min and then cooled slowly) in 100 mM KCl
solution.
Circular Dichroism Spectroscopy. Next, we performed CD
spectral titrations involving individual ligands with the
preformed G4DNA formed in LiCl buffer (10 mM sodium
cacodylate having 100 mM LiCl). No noticeable structural
change was observed with 2a and 2b based on the CD spectral
profiles (Figure S9, Supporting Information). But the positive
peaks in the CD spectra at 245 and 295 nm became less
intense, indicating some interaction of G4DNA with the
ligands. With dimeric ligands having longer linker, i.e., 2c and
2d, the peak at 295 nm was found to be much less pronounced,
suggesting their stronger interactions with G4DNA (Figure
S10, Supporting Information).
the planar G-quartet regions of the G4DNAs, there are also
several grooves available for the interaction with a guest
molecule.34 Incidentally, we did not observe any isodichoric
point in the CD spectral titrations, which could be due to the
presence of more than one G-quadruplex conformations in
solution. This was also consistent with the reported NMR
spectrum of the 22-mer human telomeric sequence in K+
solution. The NMR spectrum indicated existence of a broad
envelope with some fine lines, implying the presence of
multiple conformational isomers of the G4DNA.13a An induced
CD signal (ICD) appeared near 330 nm above the [ligand]:
[DNA] ratio of 15, which could be due to the interaction of the
added ligand with the chiral grooves of the G4DNA. The side
chains of the G4DNA binding compounds also have their role
in groove binding.35 Benzimidazole molecules are known to
cause DNA aggregation and have been shown to undergo self-
aggregation at higher concentrations.27,36 At higher [ligand]:
[DNA] ratio (particularly above r = 25), the ICD signal
increased significantly and merged with the peak at 295 nm.
Such an observation at high compound concentration is
Solution structure of K+ stabilized G-quadruplex has
significant biological relevance.13d,14 Hence, we performed
CD spectral titrations of preformed G4DNA in K+ solution
with 2d. The peaks observed at 250, 270, and 292 nm in the
CD spectra were a bit more intense and the negative band
minimum around 232 nm started going up at lower
concentrations of 2d (Figure 4A) although the conformation
of the G4DNA appeared to have remained invariant. Besides
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possibly due to the formation of a distorted structure or owing
to some pronounced aggregation of the ligands.27
7A, Table 1). We have chosen LiCl solution because Li+ folds
telomeric DNA into (like Na+) G4DNA but it does not
stabilize the G4DNA structure on its own.32 Therefore, this
study allows one to have a good idea about the stabilization
provided by any G4DNA interacting ligand. Electron-donating
substituents in the ligands that interact with the G4DNA play
an important role in the G4DNA stabilization. Many of the
established G4DNA interacting ligands, e.g., berberine,38
telomestatin,39 dounomycin,40 adrinamycin,41 etc., contain
functional groups with electron-donating character. Side chains
of the G4DNA-specific ligands may also play an important role
in the G4DNA stabilization.27,35 Ligand 1b, having an electron-
donating ethoxy group at the phenyl ring and 2-hydroxyethyl
group at the piperazine units, provide higher stabilization to the
G4DNA than that of 1a (Table 1), which possess an ethoxy
group at the phenyl ring but only a methyl group at the
piperazinyl rings. The benzimidazole hydrogens in such ligands
may also play their role in the structural transition and
consequent stabilization of the G4DNA.
When added to the preformed G4DNA in LiCl solution at
[ligand]:[DNA] ratio = 5, the dimeric ligands 2c and 2d
induced lesser stabilization (Table 2). The melting curves were
not so cooperative (Figure S11, Supporting Information). This
might be because of a complex formation between the ligand
(having oligo-oxyethylene linker) and Li+ ion, which was also
evident from the fluorescence studies.
But interestingly, when the G4DNA was formed in presence
of 2d in LiCl solution (first heated at 90 °C for 5 min, and then
allowed to cool slowly), it showed higher stabilization (13 °C,
Table 2) with a characteristic melting curve (Supporting
Information). The ligand 2d might be able to occupy every
possible binding site when single-stranded Hum21 starts folding
in presence of the ligand into a G-quadruplex structure, leading
a higher stabilization.
But dramatic changes occurred in the CD spectral profile of
the G4DNA formed in presence of 2d in 100 mM K+ solution.
At [ligand]:[DNA] ratio = 5, the small positive peak at 245 nm
in the CD spectra disappeared with the concomitant
appearance of a shoulder and instead the small peak at 270
nm became more intense while the peak at 295 nm became less
pronounced (Figure 4B). The shallow at 232 nm also became
red-shifted to 237 nm. Interestingly, at [ligand]:[DNA] ratio of
10, all the three peaks merged to give a peak at 265 nm and a
shoulder at 295 nm (Figure 4B). This profile is similar to the
CD spectral profile of the parallel G-quadruplexes formed by
human and many nonhuman telomere sequences.16,37 The only
difference is the presence of a shallow near 237 nm and not a
negative peak. At higher [ligand]:[DNA] ratio (at [ligand]:
[DNA] ratio = 50, i.e., 200 μM of 2d), a distorted structure or
pronounced aggregation was observed as it was in the case of
the preformed G4DNA. We also examined the effect of
monomer 1b on the conformation of Hum21 quadruplex in 100
mM K+ solution (preformed G4DNA and G4DNA formed in
the presence of the compound) and obtained similar results as
described in the case of 2d (Figure 5).
These results suggest that the compounds 1b and 2d are
capable of interacting with the preformed K+-quad without
causing any structural alteration. But these ligands are capable
of directing the folding of randomly structured Hum21
sequence into the parallel-stranded G4DNA at low concen-
trations when the G-quadruplex is formed in 100 mM K+
solution in presence of such compounds. Similar structural
change was not, however, observed when G-quadruplex DNA
was formed in 100 mM Na+ solution in presence of 1b and 2d
(Figure 6).
Thermal Denaturation. In thermal denaturation experi-
ments, 1a and 1b showed characteristic melting curves upon
monitoring the changes in the CD spectral maximum at 295
nm as a function of temperature. These melting profiles suggest
significant stabilization (of 9.5 and 11.5 °C, respectively) of the
preformed Hum21 G4DNA in 100 mM LiCl solution at
[ligand]:[DNA] ratio of 5 upon binding with 1a or 1b (Figure
In 100 mM Na+ solution, the Hum21 G4DNA has higher
melting temperature (57 °C, Table 3) because of the
stabilization provided by the Na+ ions. To see the effect of
ligands on the stability of the G4DNA in Na+ solution, we used
a higher [ligand]:[DNA] ratio of 10. Ligand 1b and 2d caused
melting temperature increments of 11 and 14 °C respectively in
Na+ solution when monitored at 295 nm using CD spectros-
copy as a function of temperature (Figure 7B, Table 3).
Taken together, the CD titrations and the thermal
denaturation data indicate that although Li+ and Na+ ions are
capable of forming complexes with the dimeric ligands (as
evident from the fluorescence titrations), these ligands are able
to bind strongly with the Hum21 G4DNA in the presence of
such alkali metal ions, leading to the stabilization of the
G4DNA. The binding is more favorable when the G-
quadruplex structure was formed in presence of the dimeric
ligands.
CD melting results were even more striking when performed
in 100 mM K+ solution. In fact, K+ solution is biologically more
relevant and K+ ions form very stable G4DNA structures.13−16
It is therefore difficult to measure the stabilization provided by
a weak stabilizing ligand in K+ solution. At [ligand]:[DNA]
ratio =10, 1b and 2d provided less stabilization (6 and 9 °C,
respectively, Figure 8, Table 4) with the preformed G4DNA in
presence of K+ ions. Stabilization was, however, significantly
higher when the G4DNA was formed in presence of either
ligand 1b or 2d at [ligand]:[DNA] ratio of 10 in 100 mM K+
solution (14.5 and 16 °C, respectively, Figure 8, Table 4).
Figure 6. CD spectral profiles of the Hum21 (2 μM strand
concentration) G4DNA without any ligand (solid line) and G-
quadruplex DNA formed in presence of 20 μM of 1b (dashed−dotted
line) and 2d (dashed line) (first heated at 90 °C for 5 min and then
cooled slowly) in 100 mM NaCl solution.
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Figure 7. CD melting profiles (at 295 nm) of the preformed Hum21 G4DNA (2 μM, solid lines) and its complex with 10 μM of 1a, (dashed−dotted
line) and 1b, (dashed line) respectively in LiCl buffer (10 mM sodium cacodylate having 100 mM LiCl and 0.1 mM EDTA, pH 7.4). (B) CD
melting profiles (at 295 nm) of the preformed Hum21 G4DNA (2 μM, black) and its complex with 20 μM of 1b, (dashed−dotted line) and 2d,
(dashed line) in NaCl buffer (10 mM Tris-HCl buffer having 100 mM NaCl and 0.1 mM EDTA, pH 7.4), respectively.
Table 2. Thermal Denaturation Temperatures (on the basis
of DNA melting as probed by CD spectra) of Hum21
G4DNA (2 μM strand concentration) Formed in LiCl Buffer
(10 mM sodium cacodylate having 100 mM LiCl and 0.1
mM EDTA, pH 7.4), and G4DNA Complexed Either with 10
μM ([ligand]:[DNA] ratio = 5) of the Indicated Ligand at
295 nm
evidence of hysteresis and the cooling curves were not
superimposable with the heating curves. Reformation of
G4DNA was more significant in case of 2d.
Electrophoresis. To examine whether these benzimidazoles
trigger any topology change of the G-quadruplex DNA, we
performed the electrophoresis of the 5′-32P-end labeled
preformed Hum21 G-quadruplex DNA in 100 mM KCl and
G-quadruplex formed in the presence of either of 1b or 2d at
different [ligand]:[DNA] ratios in 100 mM KCl solution.
Under comparable conditions, electrophoresis was also
performed using a dT20 marker. All the G4DNA bands formed
expectedly showed considerably higher mobility than that of
the dT20 ODN under identical conditions (Figure S12,
Supporting Information). The preformed G4DNA complexed
with 1b displayed a similar mobility as that of the uncomplexed
G4DNA, suggesting that there was no change in the topology
of the G-quadruplex upon binding with 1b. But the G4DNA
formed in presence of either 1b or 2d have lower mobility than
that of the preformed G4DNA, although the former possessed
higher electrophoretic mobility than that of the dT20 ODN
(Figure S12, Supporting Information). The gradual mobility
shifts at higher [ligand]:[DNA] ratio in the case of 2d may be
due to the binding effect because 2d has higher molecular
weight (1323.975) than that of 1b (MW = 610.34). However,
the mobility of the 2d bound G-quadruplex DNA is still higher
than that of the dT20 ODN in any case. These results are
consistent with those reported earlier for the G4DNA formed
by Hum21.16 In the reported instance, both the hybrid G4DNA
and the parallel G4DNA structure formed in molecularly
crowded conditions (simulated by PEG in KCl solution) had
higher mobility than that of the dT21 marker and the parallel
G4DNA had the intermediate mobility between the hybrid
G4DNA and the dT21 marker. Hence we believe that the
mobility shifts observed in the case of G4DNA formed in
presence of either 1b or 2d are due to the structural conversion
and not due to any aggregation. If it would have been due to
aggregation, then the mobility of the ligand−DNA complex
would be much lower than that of the dT20 ODN. Also, as
suggested by the CD spectroscopic data, the aggregation, if it
occurs at all, would appear only beyond [ligand]:[DNA] ratio
>25.
a
b
entry
system
Hum21
Tm °C
ΔTm °C
1
2
3
4
5
6
37.5
47
Hum21 + 1a
Hum21 + 1b
Hum21 + 2c
Hum21 + 2d
Hum21 + 2d
9.5
11.5
7.5
8
49
45
45.5
50.5
c
13
a
The results are average of three experiments and are within 0.5 °C
b
of each other. ΔTm values were obtained from the difference in
melting temperatures of the ligand bound and uncomplexed G4DNA.
c
For entry 6, G4DNA structure was formed in LiCl buffer (first heated
to 90 °C for 5 min and then cooled slowly).
During the cooling event in KCl solution, Hum21 DNA alone
did not form any specific DNA structure (Figure 8C). Cooling
of the solution of Hum21 in the presence of either 1b or 2d
resulted in G-quadruplex formation. However, there was
a
Table 3. Melting Temperatures of the Hum21 G4DNA
Formed in NaCl Buffer and G4DNA Complexed with 20 μM
of Indicated Ligands at ([ligand]:[DNA] ratio = 10)
b
entry
system
Hum21
Tm °C
ΔTm °C
1
2
3
57
68
71
Hum21 + 1b
Hum21 + 2d
11
14
a
Changes in the circular dichroism spectral peak at 295 nm observed
as a function of temperature using 2 μm stand concentration of DNA
in 10 mM Tris-HCl, pH 7.4, having 100 mM NaCl and 0.1 mM
b
EDTA. ΔTm values were obtained from the difference in melting
temperatures of the ligand bound and uncomplexed G4DNA. The
results are average of three experiments and are within 0.5 °C of each
other.
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Table 4. Melting Temperatures of the Hum21 G4DNA (2
μM) Formed in KCl Buffer (10 mM Tris-HCl having 100
mM KCl and 0.1 mM EDTA) and G4DNA Complexed with
20 μM ([ligand]:[DNA] ratio = 10) of the Indicated
a b
,
Ligands
a
entry
system
Hum21
Tm °C
ΔTm °C
1
2
3
4
66
Hum21 + 1b
Hum21 + 1b
Hum21 + 2d
Hum21 + 2d
72
6
14.5
9
c
80.5
75
c
5
82
16
a
Melting temperatures were obtained upon following temperature
induced changes in the CD spectral peaks at 292 nm for the preformed
G4DNA and at 265 nm for the G4DNA formed in presence of a
ligand. The results are average of three experiments and are within
b
0.5 °C of each other. ΔTm values were obtained from the difference
c
in the Tms of the ligand bound and the uncomplexed G4DNA. For
entries 3 and 5, G4DNA was formed in KCl buffer in the presence of
indicated ligand (first heated to 90 °C for 5 min and then cooled
slowly).
of the above compounds in solution containing K+ ion. The
pharmacophore unit of the compounds might be tightly bound
at each binding site to provide the higher stabilization.
Telomerase Inhibition (TRAP Assay). Because telomerase is
an important drug target for treating cancers, we have examined
each ligand toward its ability to inhibit human telomerase in a
modified cell-free telomerase repeat amplification protocol
(TRAP) test.42 Ligands were tested at the concentration
ranging from 2.5 to 60 μM for effecting inhibition of the
telomerase activity. As expected, dimeric ligands were found to
be more potent toward telomerase inhibition than the
corresponding monomeric ligands (Figure 9 and Figure S13,
Supporting Information). While the monomeric ligand 1b
inhibits the telomerase activity at ∼30 μM concentration, the
dimeric compound 2d has inhibited the telomerase activity only
at 5 μM concentration (Figure 9). The IC50 values for the
telomerase inhibition have been determined and are shown in
Table 5. Interestingly, 1a showed a lower IC50 value than that
of 1b. Dimeric ligands displayed much lower IC50 values than
the monomeric ligands.
Cancer Cell-Specific Toxicity. To check the selective toxicity
of the synthesized ligands toward cancer cells, we treated 1b
and 2d with human breast cancer cells (MBA-MD231) and
normal human keratinocyte cells (HaCat) for 6 and 48 h using
MTT based cell viability assay (Figure 10). Both the ligands
showed concentration-dependent cytotoxity toward the cancer
cells. In contrast, these ligands were significantly less toxic
toward the normal cells. Observation of higher cytotoxicity of
the ligands toward the cancer cells over the normal cells may be
correlated with their ability to fold the longer telomeric ends
(in cancer cells) into the G4DNA and to inhibit telomerase in
turn.3−8 Telomerase is not believed to be active in the normal
cells, and hence they were less affected by the ligands.4,7
Molecular Modeling. To gain insight into the nature of
binding of the monomeric ligand 1b with the telomeric
G4DNA, we adopted an approach that combined molecular
docking and MD simulations. The parallel propeller-type X-ray
G-quadruplex DNA structure (PDB 1KF1)12 was a used as
template for the modeling studies. The propeller shaped
G4DNA structures could be characterized by two external G-
quartet planes, the 5′-G-quartet surface is relatively more
hydrophobic for favoring π-stacking interactions whereas the 3′-
Figure 8. CD melting profiles of the Hum21 G4DNA alone (2 μM,
solid line, at 292 nm) and its complex with 20 μM of 1b (dashed−
dotted line, A) and 2d (dashed line, B), respectively, in KCl buffer (10
mM Tris-HCl having 100 mM KCl and 0.1 mM EDTA, pH 7.4)
followed at 292 nm as a function of temperature. The dashed−dotted
curve (at 292 nm) is obtained when the ligand is mixed with a
preformed G4DNA and the dashed curve (at 265 nm) is obtained
when G4DNA is formed in presence of the ligand (first heated at 90
°C for 5 min and then cooled slowly). (C) Cooling curves of the
solutions of Hum21 DNA and its complexes with 1b and 2d.
Taken together, the CD spectroscopic and electrophoretic
mobility shift assay data illustrate how the compounds 1b and
2d direct the folding of d[G3(T2AG3)3] into parallel quadruplex
DNA structure when the G4DNA is formed in presence of each
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presence of an easily accessible groove, 1b also binds to the
grooves of the G4DNA, which is also evident from the
occurrence of an ICD signal in the CD spectra (Figure 11).
The core phenyl ring and the benzimidazole linkers that form
the central pharmacophore of the ligand 1b interact strongly
with the guanine tetrads by maximizing the π-stacking
interactions. The planar bisbenzimidazole moiety effectively
stacks over the surface of the guanine residues of one side of
the G-tetrad (Figure 11). Another possible reason for the
strong stabilization potential of these ligands might be because
of the comparable size of the bisbenzimidazole core of each
ligands with that of the G-quartet.26,27 Therefore, the molecular
feature of the central core (internal hydrogen bonds) and the
electronic/electrostatic properties make these ligands almost
perfectly suitable for the recognition of the G4DNA. Piperazine
substituted side chains go toward the grooves and make
hydrogen bonding interactions with the sugar and the
phosphate backbones of the G4DNA organization effective.
Thus one may hypothesize that the dimeric ligands, particularly
the ones having longer flexible linker, would be more effective
for the stabilization of telomeric G4DNA.
Figure 9. Representative experiments for the determination of the
telomerase inhibitory properties by the bisbenzimidazole derivatives.
TRAP assay performed with increasing concentrations 1b and 2d.
Lane 1, (−) ve control (absence of enzyme and ligand); lane 2, R =
PCR control; lanes 3, 4, 5, and 6, TRAP reaction mixture + (5, 10, 30,
and 60 μM of 1b); lanes 7, 8, 9, and 10, TRAP reaction mixture + (2.5,
5, 10, and 30 μM of 2d); lane 11, (+) ve control (absence of the
ligand). Note: TRAP assays with more ligands are given in the
Supporting Information.
CONCLUSIONS
■
In this report, we describe the design and synthesis of six new
bisbenzimidazole based ligands which efficiently stabilize the
G4DNA assembled from a human DNA telomeric sequence.
This occurs in both cases, i.e., with the preformed G4DNA and
the G4DNA formed in the presence of each ligand in solution
containing various monovalent alkali metal salts. The above
ligands direct the folding of telomeric DNA into an unusually
stable parallel-stranded conformation (as evident from the CD
melting data) when the G4DNA is formed along with the
compound in K+ solution. Dimeric ligands provide significantly
higher stabilization to the G4DNA than the corresponding
monomeric ligands in both the cases. Recent studies have
shown that 48-mer sequence d(TTAGGG)8 forms a dimeric
DNA structure containing 2-folded G-quadruplex units.45 In
this context, the dimeric compounds and their multimeric
forms, having sufficiently long intervening spacer between each
monomeric unit, should be appropriate even for the
stabilization of the G4DNA in vivo. The G4DNA structures
in human genome may be readily folded and stacked end-to-
end to form compact-stacking structures for multimers in the
elongated telomeric DNA,46 and these types of dimeric ligands
may bind with two (or more) consecutive G-quadruplex loops.
Telomerase inhibition assay reinforces this fact as dimeric
ligand (2d) inhibits the enzyme at significantly lower
concentration compared to its monomeric counterpart (1b).
We have reported earlier that the monomeric ligands exhibit
weak binding with the duplex DNA.26,27 The dimeric ligand 2d
also shows ∼100-fold higher affinity for the G4DNA than the
duplex DNA. Importantly, ligands are selectively more
cytotoxic toward cancer cells than normal cells. Higher
selectivity toward the cancer cells may be linked with the
telomerase inhibition capability of the ligands.
Table 5. IC50 Values against Telomerase Found for the
a
Different Ligands
entry
ligand
IC50 (μM)
1
2
3
4
5
6
1a
1b
2a
2b
2c
2d
22.3
28.0
10.8
6.2
6.0
5.5
a
The results are average of three experiments and are within 1% of
each other.
G-quartet surface is more favored for electrostatic interactions,
and both the 5′- and 3′- ends are potential binding sites for the
above ligands. There are four equivalent phosphate grooves
created by three TTA loops on the side, and these are quite
deep and easily accessible for the ligands.12
Molecular docking studies were first carried out to predict
the nature of possible interactions between the ligands and the
G4DNA. Previously, it was shown that ligands prefer to stack at
the top of the terminal G-tetrads.43 The intercalation of a ligand
in between the successive G-tetrads in a G4DNA is also
energetically not favorable.44 Results from our docking studies
suggest that besides having end-stacking interactions, flexible
ligands could be readily accommodated in the groove regions of
the propeller-type structures.
On the basis of the docking results, we have taken the lowest,
final docked energy ligand−G4DNA complexes. Then MD
simulations (8 ns) were performed on four complexes formed
by the G4DNA (propeller-type) with ligand 1b in two binding
modes (end-stacking and groove-binding). Each model was
found to be quite stable during the dynamics runs. Monomeric
ligand 1b was found to stack on the 5′-side. Because of the
In summary, we have designed a new class of G-quadruplex
stabilizing ligands and demonstrated that oligo-oxyethylene
linkers of suitable length form a part of a pharmacophore
capable of having favorable interactions with the G-tetrads. We
have synthesized a first generation of monomers which show
excellent affinity and selectivity toward the G-quadruplex.
Moreover, we have demonstrated that dimerization of ligands
via appropriate choice of linker and its optimal length has
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Figure 10. Effect of ligands 1b and 2d on the cell viability after short-term exposure (for 6 and 48 h) of human breast cancer cells (MBA-MD231)
and normal human keratinocyte cells (HaCat) at specified concentrations as measured by the MTT assay. Each experiment was performed three
times and an average at each point is shown.
Figure 11. (A) Optimized structure at the B3LYP/6-31G* level of theory of 1b and (B) chemical structure of the G-tetrad with interatomic
distances as given in the crystal structure in ref 12. Simulated structure of the 1b−G4DNA complex: (C) 1b stacking on the surface of G-quartet
plane at 1:1 ratio. (D) 1b bound to the groove of the propeller shaped G4DNA. K+ ions that come from the bulk solution, stabilize the complex
during dynamic run, are shown by purple stars. Ligand is shown in “sticks” model colored by atom type (purple−white). Propeller shaped G4DNA is
represented as a cartoon form (green−orange).
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(calcd for C68H82N16O5·1.5H2O): C, 67.25; H, 7.05; N, 18.45. Found:
C, 67.12; H, 6.95; N, 18.42.
applicability and relevance in the field of G-quadruplex ligand
discovery. These findings should spearhead a new modular
approach for the identification and synthetic attainment of lead
structures, analogues, and modification of existing quadruplex
binders. This strategy should lead to increased diversity and, in
turn, selectivity and potency necessary for the anticancer drug
design.
1,11-Bis-[2,2′-(5-phenoxy-1,3-phenylene)-bis[5-(4-(2-hydroxyeth-
yl)-1-piperazinyl)-1H-benzimidazole]]-3,6,9-trioxo-undecane (2d).
Freshly prepared 4-[4-(2-hydroxyethyl)-piperazin-1-yl]-benzene-1,2-
diamine (14, 188 mg, 0.8 mmol) and 12 (91 mg, 0.2 mmol) and
Na2S2O5 (75 mg, 1.6 mmol) were reacted as per the general protocol
given above. Isolated yield 163 mg, 62%; mp > 290 °C. IR: 3410, 3016,
2878, 1614, 1565, 1450, 1220 cm−1. 1H NMR (DMSO-d6) δ 13.1 (bs,
4H), 7.85 (bs, 4H), 7.5 (bs, 6H), 7.0 (bs, 8H), 4.3 (bs, 4H), 3.8 (bs,
12H), 3.68 (bs, 16H), 3.58 (bs, 24H), 3.2 (bs, 16 H). 13C NMR
(DMSO-d6) δ: 159.19, 158.79, 150.24, 146.50, 132.09, 119.53, 116.95,
115.50, 114.24, 113.01, 79.24, 69.83, 68.86, 67.72, 57.88, 55.4, 51.51,
47.06, 42.0. m/z (MALDI-TOF) found, 1323.975 (calcd, 1323.978,
[M + H]+). Anal. (calcd for C72H90N16O9·1.5H2O): C, 64.03; H, 6.94;
N, 16.59. Found: C, 63.98; H, 7.0; N, 16.5.
MATERIALS AND METHODS
■
Materials and General Spectroscopic Characterizations. See
Supporting Information. All tested ligands were found to be at least
>95% pure by elemental analysis (Table S1, Supporting Information).
Synthesis. Synthesis of precursors (5−12) has been described in
the Supporting Information.
General Method for the Synthesis of 1,3-Phenylene-bis-
(piperazinyl benzimidazole) Derivatives (2a−2d). To a freshly
prepared ethanolic solution of either 4-(4-methylpiperazin-1-yl)-
benzene-1,2-diamine (13)27 or 4-[4-(2-hydroxyethyl)piperazin-1-yl]-
benzene-1,2-diamine (14),27 1/4th equivalent of the appropriate
tetraaldehyde (either 11 or 12, Supporting Information) was added,
and to this solution, two equivalents of sodium metabisulphite
(Na2S2O5) dissolved in minimum quantity of water was added. The
resulting reaction mixture was refluxed for 8 h with stirring, then
cooled to room temperature, and finally filtered through Celite.
Ethanol was then removed from the reaction mixture under reduced
pressure to obtain a residue. This material was then purified by column
chromatography (EtOAc/MeOH) on silica gel (70−220 mesh size) to
obtain the required product as given in the following.
1,8-Bis-[2,2′-(5-phenoxy-1,3-phenylene)-bis[5-(4-methyl-1-piper-
azinyl)-1H-benzimidazole]]-3,6-dioxo-octane (2a). Freshly prepared
4-(4-methylpiperazin-1-yl)-benzene-1,2-diamine (13, 165 mg, 0.8
mmol) and 11 (83 mg, 0.2 mmol) and Na2S2O5 (75 mg, 1.6 mmol)
were reacted as per the general protocol. Isolated yield 138 mg, 60%;
mp 287−288 °C. IR: 3356, 2956, 2835, 2729, 1633, 1455, 1223 cm−1.
1H NMR (DMSO-d6) δ 13.2 (bs, 4H), 7.9 (bs, 4H), 7.6 (bs, 6H), 7.0
Oligonucleotides. HPLC purified oligodeoxyribonucleotides
(ODNs) d[G3(T2AG3)3], abbreviated as Hum21, and d[T20] were
purchased from Sigma Genosys, Bangalore. Their purity was
confirmed using high resolution sequencing gel. The molar
concentration of each ODN was determined from absorbance
measurements at 260 nm based on their molar extinction coefficients
(ε260) 215000 and 148400, respectively, for d[G3(T2AG3)3] and
d[T20], respectively.
G-Quadruplex Formation, Circular Dichroism Spectroscopy,
Tm Studies, Fluorescence Spectroscopy, UV−Vis Titrations,
Polyacrylamide Gel Electrophoresis, Telomerase Assay, and
Computational Studies. These studies have been performed as
described in our earlier reports.26,27
Cell Viability Assay. Human breast cancer cells (MBA-MD231)
and normal human keratinocyte cells (HaCat) were seeded in 96-well
plates (15.0 × 103/well). Cells were grown for 24 h before treatment
to get >70% confluency and exposed to various concentrations of
ligands in presence of 0.2% FBS. After either 6 or 48 h incubation at
37 °C in a humidified atmosphere of 5% CO2, old medium was
replaced with the new one containing 10% FBS in DMEM and cells
were further grown for 42 h post treatment. Then 20 μL of 5 mg/mL
methyl thiazolyl tetrazolium (MTT) reagent was added to 200 μL of
the medium present in each well, and cells were further incubated for 4
h. Old medium was discarded, and formazan crystals were dissolved in
DMSO and reading (fluorescence intensity, FI) was taken at 595 nm
in the ELISA plate reader. All ligand doses were parallel tested in
triplicate. Percentage cell viability was calculated by using the formula,
(bs, 8H), 4.3 (bs, 4H), 3.7 (bs, 24H), 3.05 (bs, 16H), 2.65 (bs, 12 H).
13C NMR (DMSO-d6) δ: 159.29, 149.31, 147.4, 141.5, 139.6, 136.0,
132.2, 128.16, 118.9, 116.8, 114.5, 113.7, 111.2, 105.3, 103.0, 97.3,
69.0, 67.5, 58.13, 53.9, 49.5, 48.8, 42.28. m/z (MALDI-TOF) found,
1159.610 (calcd, 1159.609, [M + H]+). Anal. (calcd for
C66H78N16O4·H2O): C, 67.32; H, 6.85; N, 19.03. Found: C, 67.25;
H, 6.87; N, 18.95.
%cell viability = [(FI
of treated cells
(595)
1,8-Bis-[2,2′-(5-phenoxy-1,3-phenylene)-bis[5-(4-(2-hydroxyeth-
yl)-1-piperazinyl)-1H-benzimid-azole]-3,6-dioxo-octane (2b).
Freshly prepared 4-[4-(2-hydroxyethyl)-piperazin-1-yl]-benzene-1,2-
diamine (14, 188 mg, 0.8 mmol) and 11 (83 mg, 0.2 mmol) and
Na2S2O5 (75 mg, 1.6 mmol) were reacted as per the general protocol
given above. Isolated yield 158 mg, 62%; mp > 290 °C. IR: 3404, 3019,
2870, 1634, 1570, 1456, 1218 cm−1. 1H NMR (DMSO-d6) δ 13.2 (bs,
4H), 7.9 (bs, 4H), 7.5 (m, 6H), 7.0 (bs, 8H), 4.25 (bs, 4H), 3.72 (bs,
20H), 3.15 (bs, 16H), 2.6 (bs, 24 H). 13C NMR (DMSO-d6) δ:
159.31, 149.35, 143.7, 142.1, 141.7, 139.8, 136.1, 128.16, 120.4, 113.5,
107.5, 103.1, 97.1, 70.0, 67.6, 59.95, 58.15, 53.1, 49.60, 48.02, 42.68.
m/z (MALDI-TOF) found, 1279.705 (calcd, 1279.682, [M + H]+).
Anal. (calcd for C70H86N16O8·2H2O): C, 63.91; H, 6.90; N, 17.04.
Found: C, 63.78; H, 6.92; N, 16.93.
1,11-Bis-[2,2′-(5-phenoxy-1,3-phenylene)-bis[5-(4-methyl-1-pi-
perazinyl)-1H-benzimidazole]]-3,6,9-trioxo-undecane (2c). Freshly
prepared 4-(4-methylpiperazin-1-yl)-benzene-1,2-diamine (13, 165
mg, 0.8 mmol) and 12 (91 mg, 0.2 mmol) and Na2S2O5 (75 mg,
1.6 mmol) were reacted as per the general protocol given above.
Isolated yield 146 mg, 61%; mp 285−286 °C. IR: 3352, 2959, 2838.5,
2730, 1630, 1450.5, 1227 cm−1. 1H NMR (DMSO-d6) δ 11.4 (bs, 4H),
8.0 (bs, 4H), 7.5 (m, 6H), 7.0 (bs, 8H), 4.3 (bs, 4H), 3.8 (bs, 28H),
3.2 (bs, 16H), 2.9 (bs, 12 H). 13C NMR (DMSO-d6) δ: 159.31, 158.8,
150.2, 148.44, 147.34, 139.12, 129.33, 129.06, 116.95, 115.50, 99.89,
99.12, 79.22, 69.88, 68.78, 67.93, 52.11, 49.02, 46.54, 41.83. m/z
(MALDI-TOF) found, 1203.892 (calcd, 1203.894, [M + H]+). Anal.
− FI
of plain DMSO)
(595)
/(FI
of untreated cells
(595)
− FI
of plain DMSO)] × 100
(595)
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of the precursors for the dimeric ligands, NMR
spectra of the key compounds, additional CD and fluorescence
data, computational results, CD melting curves, topology
change in Hum21 by electrophoresis, additional TRAP assays
of ligands. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (91)-80-22932664. Fax: (91)-80-22930529. E-mail:
sb@orgchem.iisc.ernet.in.
Notes
The authors declare no competing financial interest.
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parallel quadruplexes from human telomeric DNA. Nature 2002, 417,
876−880.
Note: “preformed” G-quadruplex DNA refers to the G-
quadruplex DNA formed in absence of any added ligand (i.e.,
the unstructured DNA is annealed in the chosen buffer, having
indicated salt, however, with no added ligand). [ligand]:[DNA]
ratio = equiv of ligand (μM) with respect to that of DNA (μM
of DNA strand). [DNA]:[ligand] ratio = equiv of DNA (μM of
DNA strand) with respect to that of ligand (μM).
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ACKNOWLEDGMENTS
■
This work was supported by a grant (J.C. Bose Fellowship grant
to Prof. S. Bhattacharya) from the Department of Science and
Technology (DST), New Delhi, India. A.K.J. is thankful to
DST for a grant as a Fast Track Project and DBT New Delhi
for a postdoctoral fellowship. We thank Santosh K. Misra for
technical assistance.
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ABBREVIATIONS USED
■
G4DNA, G-quadruplex DNA; ODN, oligodeoxynucleotide;
CD, circular dichroism; ICD, induced circular dichorism; PEG,
polyethylene glycol; TRAP, telomerase repeat amplification
protocol; FI, fluorescence intensity; EDTA, ethylenediaminete-
traacetic acid; MTT, methyl thiazolyl tetrazolium; DMSO,
dimethyl sulphoxide; NMR, nuclear magnetic resonance;
MALDI, matrix assisted laser desorption ionization; IR, infrared
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