Conjugation of a 3-(1H-phenanthro[9,10-d]imidazol-2-yl)-1H-indole intercalator to a triplex oligonucleotide and to a three-way junction

Article abstract of DOI:10.1016/j.bmc.2011.11.013

A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was synthesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction with phenanthrene-9,10-dione in the presence of

Full text of DOI:10.1016/j.bmc.2011.11.013

Bioorganic & Medicinal Chemistry 20 (2012) 207–214
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Conjugation of a 3-(1H-phenanthro[9,10-d]imidazol-2-yl)-1H-indole
intercalator to a triplex oligonucleotide and to a three-way junction
Maha I. Fatthalla a,b, Yehya M. Elkholy b, Nermeen S. Abbas b, Adel H. Mandour c,
Per T. Jørgensen a, Niels Bomholt a, Erik B. Pedersen a,
⇑
a Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
b Department of Chemistry, Faculty of Science, Helwan University, 11795 Ain Helwan, Cairo, Egypt
c Natural Product Chemistry, National Research Center, Dokki, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2011
Revised 2 November 2011
Accepted 7 November 2011
Available online 13 November 2011
A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was syn-
thesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction
with phenanthrene-9,10-dione in the presence of ammonium acetate to form a phenanthroimidazole moi-
ety linked to the indole ring. Insertion of the new intercalator as a bulge into a Triplex Forming Oligonucleo-
tide resulted in good thermal stability of the corresponding Hoogsteen-type triplexes. Molecular modeling
supports the possible intercalating ability of M. Hybridisation properties of DNA/DNA and RNA/DNA three-
way junctions (TWJ) with M in the branching point were also evaluated by their thermal stability at pH 7.
DNA/DNA TWJ showed increase in thermal stability compared to wild type oligonucleotides whereas this
was not the case for RNA/DNA TWJ.
Keywords:
Indole
Triplex Forming Oligonucleotide
Three-way junction
Thermal stability
Fluorescence
Ó 2011 Elsevier Ltd. All rights reserved.
Molecular modeling
1. Introduction
selectively to tumor cells.8 Fully phosphorothioate 25-mer antipar-
allel TFO shows significant results to attenuate liver fibrosis.9 Inter-
Secondary structure formation, including triple helix, is one of
the extraordinary properties that nucleic acid adopts. The third oli-
gonucleotide strand was first described by Felsenfeld and Rich,1 re-
ferred to as the Triplex Forming Oligonucleotide (TFO). The triplex
can be formed if the targeted strand is rich in purines, which in
addition to the bonds of the Watson–Crick base pairing can form
two further hydrogen bonds, Hoogsteen or reverse Hoogsteen, able
to bind with a third matching oligonucleotide strand.2 As the hu-
man genome often contains sequences rich in purines,3 specific du-
plex DNA targeting by TFO provides an attractive strategy to induce
genetic manipulation by homologous exchange using single-
stranded oligodeoxynucleosides with the ultimate goal of repairing
genetic defects (site-directed mutagenesis, site-directed recombi-
nation, etc.) in human cells.4,5 Duplex DNA targeted by TFO has also
been reported for regulating the expression of genes that are
responsible for the synthesis of protein(s)/enzyme(s) for specific
diseases by locking their transcription (antigene).6 In this context
TFO could be used to deliver drugs to a specific site in the genome7
and to direct validated anticancer drugs to genes carrying cancer-
specific mutations or translocations thereby inducing DNA damage
estingly, the TFOs were also used in antigene radiotherapy,10 for
example treating psoriasis and other inflammatory or malignant
skin diseases by psoralen conjugated to TFO.11 Also intercellular
gene targeting, identification of genes that is responsible for the cell
growth and malignant transformation,12 TFO helps to study the
molecular mechanism of enzymes by interfering binding with poly-
merase13 endonuclease14 and methylase15 enzymes, to label DNA
site-specifically and to purify and recognize DNA.16 Circular TFOs
are of particular interest, since these are not substrates for degrada-
tion by exonucleases.17
Junctions in nucleic acid structures are formed when three or
more helices meet at a single point. These branched junctions are
important intermediates in many biological functions and play
important roles in many cellular processes.18 Three-way junctions
(TWJs) are the simplest type of junctions and consist of three dou-
ble helical arms connected at the junction point. TWJs are formed
both in DNA and RNA. In RNA, they are involved in splicing19 and
translation,20 while in DNA, they are formed transiently during
DNA replication (the replication fork21) and during recombination
involving phages.22 Furthermore, DNA-TWJs were proposed to oc-
cur in the triplet repeats expansions found in genetically unstable
genomic DNA associated with human diseases such as Hunting-
ton’s.23 Beside their roles in biological processes, branched
⇑
Corresponding author. Tel.: +45 65502555; fax: +45 66158780.
E-mail address: ebp@ifk.sdu.dk (E.B. Pedersen).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.013

208
M. I. Fatthalla et al. / Bioorg. Med. Chem. 20 (2012) 207–214
junctions offer a unique window into DNA nanotechnology, which
includes the controlled self-assembly of nanometer-scale molecu-
lar fragments.24 Because TWJs are the smallest type of junction
structures, they are used as a model system to gain insight into
other complex and multi-branched junction structures, such as
the Holliday junction, which is a key intermediate in homologous
recombination. TWJs, therefore, provide a suitable system for
assessing the quantitative features of junction structures.25
between the two ring systems. Therefore, we replaced the naph-
thalene ring in the monomer Y with an indole ring system that
has different spatial arrangement. The new synthesized monomer
M was inserted into a 14-mer homopyrimidine oligonucleotide
for thermal triplex stability studies and this proved enhanced tri-
plex stability compared to wild type. In addition, DNA and RNA
TWJs hybridisation stabilities were also studied by introducing
monomer M into the junction region as a bulge. It is worth to note
that we are the first to use an indole ring as an intercalating oligo-
nucleotide system. Indoles have previously been used as artificial
DNA bases in oligonucleotides.38
Triplexes are thermodynamically less stable than the corre-
sponding duplexes at neutral pH because protonation of cytosine
is providing stable Hoogsteen base pairing in pyrimidine tri-
plexes.26 This poor stability limits its applicability in vitro and
in vivo.27 To overcome this limitation, many trials have been made
to increase binding ability either in the major or minor grooves of
the duplex. Developing TFOs that have chemically modified nucle-
otides that bind in the major groove region such as locked nucleic
acid (LNA),28 peptide nucleic acid (PNA),29 or bulge insertion of
heterocyclic compounds (intercalators)30 proofed its efficiency to
increase the affinity of the TFO for specific target sequences and
to stabilize the corresponding triplexes.31 Also some small com-
pounds like aminoglycosides32 and some polyhydroxylated com-
pounds33 function as DNA minor groove binding ligands.
Addition of metal ion as nanoparticles have been recently re-
ported.34 Our interest is to develop new intercalating moieties
for bulge insertion in the TFO. It is believed that the intercalator
design being able to stabilize the TFO via intercalation and stacking
in Hoogsteen-type helixes should contain a long and large conju-
gated aromatic surface enough to place the intercalator into the
dsDNA part of the triple helix together with a short flexible linker
to the sugar–phosphate backbone to minimize the entropy effect.35
Recently, we have reported the synthesis and properties of inter-
calating nucleic acids designed for Hoogsteen-type triplexes with
bulge insertions of aromatic moieties into the middle of homopyr-
imidine oligonucleotide (ON). An extraordinary high thermal stabil-
ity was found for the corresponding triplexes. The pyrene
dependant, (R)-1-O-[4-(1-pyrenylethynyl)phenylmeth-yl]glycerol
(W, twisted intercalating nucleic acid, TINA, Fig. 1)36 and the phen-
anthrene dependant monomers (S)-4-(4-(1H-phenanthro[9,10-
d]imidazol-2-yl)-phenoxy)butane-1,2-diol (X) and (S)-4-(4-(1H-
phenanthro[9,10-d]imidazol-2-yl)naphalen-1-yloxy)butane-1,2-
diol (Y). In the latter molecule, the phenyl group in X has been re-
placed by a naphthyl group hoping to get better stacking ability
(Fig. 1). However, a decrease in the thermal stability of Y was re-
ported compared to W and X which was explained by sterical
clashes between protons on the naphthalene and the imidazole
moieties forcing the naphthalene ring out of planarity.37
2. Chemistry
The intercalating nucleic acid monomer 9 was synthesized as
described in Scheme 1. N-Alkylation of indole-3-carboxaldehyde
is a key step in order to accomplish linking of the intercalating
moiety to a backbone monomer. Many trials were made using a
dioxolane ethanol as an alkylating agent to get a high yield of
N-alkylated indole-3-carboxaldehyde 4. Ruthenium-catalyzed
alkylation,39 Mitsunobu reaction40 and microwave assisted reac-
tion41 all failed to get the product in a higher yield than 20%. Also
in another strategy by formylation of the N-alkylated indole,42 we
failed and got the starting material as the sole product. It was more
successful to use the classical method that involve treatment of the
commercially available indole-3-carboxaldehyde 3 with NaH as a
base together with triethyl amine using the dioxolane mesylate 2
as the alkylating reagent in dry DMF.43 Hydrochloric acid was sub-
sequently added to cleave the isopropylidene protection group to
get the diol 4 in 49% overall yield. Treatment of 4 with phenan-
threne-9,10-dione 5 and ammonium acetate in hot glacial acetic
acid44 afforded the diacetylated monomer 6 in 76% yield instead
of the expected diol monomer 7. The latter was obtained in 78%
yield as a solid compound by subsequent treatment of the diacet-
ylated compound 6 with saturated methanolic ammonia.45 The
primary hydroxy group was protected by reaction with 4,40-dime-
thoxytrityl chloride (DMTCl) in anhydrous pyridine containing tri-
ethyl amine46 at room temperature under a N2 atmosphere. Silica
gel chromatography afforded the DMT-protected compound 8 in
85% yield. The secondary hydroxy group was phosphitylated over-
night with 2-cyanoethyl N,N,N0,N0-tetraisopropyl phosphorodiami-
dite in the presence of diisopropyl ammonium tetrazolide as
activator in anhydrous CH2Cl2 to afford 9 in 50% yield (Scheme
1). The obtained phosphoramidite 9 was incorporated into oligonu-
cleotides by a standard phosphoramidite protocol on an automated
DNA synthesizer. However, an extended coupling time (15 min)
was used for the modified amidite in the oligonucleotide. All mod-
ified oligodeoxynucleotides (ODNs) were purified by reversed-
phase HPLC, and confirmed by MALDI-TOF-MS analysis as reported
in Section 7. The purity of the final sequences was determined by
ion-exchange HPLC (IE-HPLC) to be more than 95%.
In the present work we decided to make some more modifica-
tion on the X monomer design in order to minimize sterical clashes
3. Thermal stability studies
The thermal stability of parallel triplexes and TWJ with bulge
insertion of the intercalating monomer M was evaluated by ther-
mal denaturation experiments using the UV melting method. The
melting temperatures (Tm, °C) were determined as the maximum
of the first derivative of melting curves.
TFO’s thermal stability was studied with the wavelength
k = 260 nm at pH 5.0, 6.0 and 7.2. An excess of the pyrimidine to
the purine strand ratio (1.5:1 mM) in the duplex was used to avoid
a disturbing melting of the parallel duplex at pH 5.0 and 6.0. The
triplex from the TFO ON2 and the duplex D1 shows high thermal
stability at pH 6.0 and pH 7.2 when compared with the wild type
ON1 (Table 1). Because of overlapping triplex and duplex meltings
N
N
NH
N
NH
NH
N
O
O
O
O
O
O
P
O
DNA
DNA
O
O
O
P
DNA
O
P
DNA
O
O
P
DNA
O
O
O
O
O
O
DNA
DNA
DNA
W
X
Y
M
Figure 1. Structure of intercalators M, W, X and Y.

M. I. Fatthalla et al. / Bioorg. Med. Chem. 20 (2012) 207–214
209
CHO
OH
a
OMs
CHO
O
O
5
N
H
3
N
d
O
O
b,c
O
O
N
N
NH
1
2
OH
4
OH
OAc
OAc
6
N
N
N
NH
f
NH
e
N
OR
OH
ODMT
OH
7
8
R=H
g
9 R=P(NiPr2)O(CH2)2CN
Scheme 1. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, 0 °C, 2 h; (b) NaH, DMF, Et3N, 55–60 °C, 72 h; (c) 1 N HCl, DMF, rt, 2 h; (d) CH3COONH4, CH3COOH glacial, 90 °C,
72 h; (e) NH3/MeOH, rt, 72 h; (f) DMTCl, pyridine, Et3N rt, 72 h; (g) N,N0-diisopropylammonium tetrazolide, 2-cyanoethyl N,N,N0,N0-tetraisopropylphosphordiamidite, rt, 48 h.
at pH 5.0, the stability of the modified and wild type triplexes were
also measured at k = 373 nm where the intercalator has a UV
absorption. When comparing the thermal melting with the previ-
ously published data37 for the phenoxy comprising intercalator X
(ON3) and the naphthoxy comprising Y (ON4) as shown in Table
1, we found a lower thermal stability of the newly modified indole
comprising M TFO even though a more favorable geometry was
anticipated to avoid sterical clashes. This is best seen at pH 6 where
the observed triplex stability was
D
Tm = 6.5 °C for the intercalator
Tm = 12 °C
M compared to Tm = 18 °C for the intercalator X and
D
D
for Y. The lower stability of the indole interalator system may be
explained by a shortening of the intercalator compared to the
naphthoxy system which may be inserted deeper into the triplex
without disturbing the backbone. Another interesting feature is
the kinetics for the formation of the triplex. Therefore heating/
annealing curves for the matched triplex ON2/D1 were recorded
at pH 5.0, 6.0 and 7.2 as shown in Fig. 2. At pH 6.0 there was found
a hysteresis profile which was similar to a previously reported
Figure 2. Melting profile of triplex melting (up and down) of ON2/D1 at different
pH’s recorded at 260 nm versus temperature difference (1 °C/min) from 10–65 °C in
20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2.
hysteresis profile for a wild-type triplex47 indicating a similarity
in the kinetics for the intercalating oligonucleotide ON2. Due to
overlapping duplex and triplex meltings at pH 5, a clear picture
of the hysteresis could not be deduced which was also the case
at pH 7.2 because of a low triplex melting temperature.
Table 1
Tm (°C) data for melting of triplexes evaluated from UV melting curves (k = 260 nm)
Entry
TFO
Parallel triplex
30-CTGCCCCTTTCTTTTTT
50-GACGGGGAAAGAAAAAA (D1)
The sensitivity to Hoogsteen mismatches were also studied for
parallel triplexes with a bulge insertion of the intercalators M, X
and Y in the TFO at pH 6.0 (Table 2, Supplementary data, Fig. S1).
X and Y was better than M to discriminate neighboring Hoogsteen
mismatches, ON3 (15–23.5 °C), ON4 (15–24 °C) compared to ON2
(10.5–16.5 °C).
Hybridisation properties of oligonucleotides ON5–7 when
targeting DNA (ON11) or RNA (ON12) forming a TWJ were eval-
uated in thermal stability studies at pH 7 (Table 3, Supplemen-
tary data, Fig. S2). TWJ was composed of a single stranded
DNA or RNA stem-loop sequence with two single stranded
regions being complementary to the oligonucleotides ON5–7
pH 5.0
pH 6.0
pH 7.2
ON1
ON2
ON3
ON4
50-CCCCTTTCTTTTTT-30
50-CCCCTTMTCTTTTTT-30
50-CCCCTTXTCTTTTTT-30
50-CCCCTTYTCTTTTTT-30
55.5a,b
55.5a,b
59.5c,d
55.0c,d
28.5a
<10.0c
15.5c
35.0a
46.5c,d
40.5c,d
26.0c,d
18.5c,d
a
C = 1.5
l
M of ON1-2, 1.5
l
M of pyrimidine strand and 1.0
of dsDNA (D1) in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 5.0
l
M of purine strand
and pH 6.0.
b
Third strand and duplex melting overlaid. Tm values confirmed at 373 nm.
C = 1.5 lM of ON1-3, 1.0 lM of each strand of dsDNA (D1) in 20 mM sodium
cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 5.0, pH 6.0 and pH 7.2.
c
d
Data taken from Ref. 37.

210
M. I. Fatthalla et al. / Bioorg. Med. Chem. 20 (2012) 207–214
Table 2
Tm (°C) data for melting of mismatched Hoogsteen parallel triplexes evaluated from UV melting curves (k = 260 nm) at pH 6.0
Entry
TFO
D1, K. L = T. A
D2, K. L = A. T
D3, K. L = C. G
D4, K. L = G. C
Sequence 30 CTGCCCCTTKCTTTTTT
50-GACGGGGAALGAAAAAA
ON1
ON2
ON3
ON4
50-CCCCTTTCTTTTTT-30
28.5a
<10.0a
18.5a
<10.0a
19.0a
<10.0a
24.5a
50-CCCCTTMTCTTTTTT-30
50-CCCCTTXTCTTTTTT-30
50-CCCCTTYTCTTTTTT-30
35.0a
46.5b,c
40.5b,c
23.0b,c
16.5b,c
29.5b,c
21.0b,c
31.5b,c
25.5b,c
a
b
c
C = 1.5
C = 1.5
Data taken from Ref. 37
l
l
M of ON1-2, 1.5
M of ON1-3, 1.0
l
M of pyrimidine strand and 1.0
lM of purine strand of dsDNA (D1) in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 6.0.
lM of each strand of dsDNA (D1) in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 6.0.
Table 3
Tm (°C) dataa for melting of DNA/RNA three-way junctionsb evaluated from UV melting curves (k = 260 nm) at pH 7.0
a
C = 1.0
l
M of each strand of DNA/RNA TWJ in 15 mM Na2HPO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0.
b
Italic sequences are bases incorporated in the stem loop.
having an intercalator M inserted at the junction in one case and
in other cases with an additional thymidine base T before or
after the intercalator M (Table 3). We suppose that TWJ has
two coaxially stacking arms and a third deflecting arm that
can be stabilized by the intercalator as a stacking lid onto that
arm at the junction site. In order to evaluate the thermal stabil-
ities of the modified TWJ we made a comparison with wild type
TWJs with none, one or two insertions of T corresponding to the
site of insertion of M. For DNA (ON11) the temperature range of
the meltings were within 2 °C when hybridised with the wild
type oligos ON8–10 whereas for the same oligos upon hybridisa-
tion to RNA (ON12), the stability increased with the number of T
ing temperature. On the other hand, when the oligos with inser-
tion of M were targeting RNA (ON12) no substantial increase in
melting temperature could be obtained when compared to the
wild type oligo with two T insertions.
The oligos ON5 and ON8 were used to confirm targeting on both
sides of the hairpins DNA (ON11) and RNA (ON12). The sensitivity
to two mismatches in one or the other arm was studied for this
purpose (Table 4). For example, the hairpin from ON13 and ON5
is destabilized by 16.5 °C compared to the corresponding hairpin
from ON11 and ON5. A similar result was found for the ON13/
ON8 hairpin when compared to the wild type ON11/ON8 hairpin
(
D
Tm = ꢀ14.5 °C). Similar results were obtained for mismatches
insertions with
D
Tm = 4.5 °C when ON10 is compared with ON8.
to the other side of the hairpin (Table 4). Also in case of DNA/
RNA TWJ, binding to both sides of the hairpin could be proven in
the same manner. The heating/annealing profile was also studied
for ON5/ON11 (Supplementary data, Fig. S3) which shows a rapid
rehybridisation of the DNA-TWJ.
For insertion of M, only, ON5 resulted in an increase of more
than 10 °C upon hybridisation to DNA (ON11) when compared
to the wild type oligos (Table 3). Additional insertion of T
(ON6 and ON7) resulted in a slightly lower increase in the melt-
Table 4
Tm (°C) dataa for melting of mismatched DNA/RNA three-way junctionsb evaluated from UV melting curves (k = 260 nm) at pH 7.0
Entry
50-GCT CAC TMT CTC CCA-30
(ON5)
50-GCT CAC TT CTC CCA-30
(ON8)
DNA
30-CGA GTG ACG CGT TTT CGC GAG AGG GT-50 (ON11)
30-CGA GTG ACG CGT TTT CGC GAG ATT GT-50 (ON13)
40.5
24.0
28.0
13.5
30-CGT TTG ACG CGT TTT CGC GAG AGG GT-50 (ON14)
18.0
<10.0
RNA
30-CGA GUG ACG CGU UUU CGC GAG AGG GU-50 (ON12)
44.0
23.0
38.5
12.5
30-CGA GUG ACG CGU UUU CGC GAG AUU GU-50 (ON15)
30-CGU UUG ACG CGU UUU CGC GAG AGG GU-50 (ON16)
21.0
17.0
a
C = 1.0 lM of each strand of DNA/RNA TWJ in 15 mM Na2HPO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0.
Italic sequences are bases incorporated in the stem loop, underlined bases are mismatched ones.
b

M. I. Fatthalla et al. / Bioorg. Med. Chem. 20 (2012) 207–214
211
4. Fluorescence properties
The insertion of the intercalator
M
into oligonucleotides
showed a characteristic fluorescence spectrum. The fluorescence
measurements on matched and mismatched triplexes were per-
formed with ON2 as the TFO whereas for TWJ the targeting oligo-
nucleotide ON5 was used. In both cases fluorescence of single
stranded oligonucleotides were also measured. In all cases, the
spectra were recorded from 340 to 600 nm at 10 °C in the same
buffer solutions as used for Tm studies.
The spectra of triplexes were measured upon excitation at
350 nm using 1.0 lM purine strand and 1.5 lM of both the pyrim-
idine strands at pH 6.0. The fluorescence intensity was stronger for
the fully matched triplex ON2/D1 compared to the single stranded
ON2. The emission intensities of the neighboring Hoogsteen mis-
matches ON2/D2 and ON2/D4 were slightly lower and slightly
higher, respectively, than for the matched ON2/D1 whereas the
Figure 4. Representative low-energy conformations of a triplex containing bulged
insertion of M produced by AMBER* calculations of the stacking showing phenan-
throimidazole between the bases of dsDNA (a) forward fused benzene moiety, (b)
backward fused benzene moiety.
emission intensity of ON2/D3 due to G/T Hoogsteen mismatch
was considerably lower, but not lower than for the single strand
ON2 (Fig. 3a). TWJ fluorescence spectra were obtained upon exci-
tation at 330 nm, using a 1.0 lM concentration of both the hairpin
strand and the modified oligonucleotide ON5 at pH 7.0. When
hybridised to DNA hairpin ON11 or to RNA hairpin ON12 the fully
matched TWJ using the modified strand ON5 showed the highest
intensity in both cases compared to the single strand ON5
(Fig. 3b and c).
5. Molecular modeling
In order to understand the intercalating properties of the new
monomer M stabilizing the triplex, we decided to examine it via
molecular modeling studies. An AMBER⁄ force field in Macro Model
9.1, molecular modeling was used to generate representative low-
energy structures of a truncated 8mer triplex with the bulge inser-
tion of the monomer M into the middle of the triplex. As it can be
seen from Figure 4a and b the positioning of the phenanthroimi-
dazole moiety in the Watson–Crick duplex is adding to the triplex
stability via p–p interaction. The presence of indolyl moiety posi-
tioned between nucleobases of the TFO make large extent of twist-
ing forcing the fused benzene ring out of plane by sterical
interaction between protons on the indole-moiety and on the
imidazole-moiety. Twisting the indolyl moiety of intercalator M
around the single bond resulted in conformations with almost
identical intercalating properties in the triplex and no optimal con-
formation could be assigned. Figure 4a and b show these two types
of conformations in which have low stackings between indolyl
moiety and up and down nucleobases. The nucleobases of the
TFO are forced to twist out of plane weakening the stacking inter-
actions and this could explain the lower thermal stability than ex-
pected. The modeling supports the thermal stability measurements
which showed a decrease in triplex stability using intercalator M in
comparison with previously prepared intercalators X and Y, clearly
demonstrating the importance of optimal
p–p-stacking
interactions.48
6. Conclusion
Figure 3. Fluorescence emission spectra of (a) ON2 comprising monomer M upon
excitation at 350 nm and pH 6.0 forming parallel triplex and mismatched triplexes.
(b) ON5 comprising monomer M upon excitation at 330 nm and pH 6.0 forming
DNA TWJ. (c) ON5 comprising monomer M upon excitation at 330 nm and pH 6.0
forming RNA TWJ.
In this study, we have synthesized new intercalating nucleic
acid monomers M, and incorporated it into oligonucleotides giving
good yields using normal oligonucleotide synthesis procedures.
Thermal melting studies showed that this intercalator was able

212
M. I. Fatthalla et al. / Bioorg. Med. Chem. 20 (2012) 207–214
to stabilize Hoogsteen-type triplexes with good discrimination of
mismatch strands compared to wild type triplexes. However,
DNA-strands containing intercalators X and Y show higher thermal
triplex stability than DNA-strands containing intercalator M. The
intercalator M increased thermal stability of DNA TWJ-type with
good discrimination of mismatch strands compared to wild type
TWJ. In case of RNA TWJ-type, the monomer M was unable to con-
tribute with any substantial.
(DMSO) d: 33.48 (CH2CH2N), 43.26 (CH2CH2N), 65.66 (CH2OH),
68.28 (CHOH), 110.95, 116.95, 120.95, 122.32, 123.39, 124.63,
136.90, 140.74 (indole), 184.35 (CHO). HRMS (ESI) m/z Calcd for
C
C
13H15NO3Na+ (MNa+) 256.11. Found: 256.105. Anal. Calcd for
13H15NO3ꢁ0.25 H2O: C, 65.67; H, 6.57; N, 5.89. Found: C, 66.10;
H, 6.38; N, 5.84.
7.1.2. (S)-4-(3-(1H-Phenanthro[9,10-d]imidazol-2-yl)-1H-indol-
1-yl)butane-1,2-diyl diacetate (6)
Phenanthrene-9,10-dione 5 (0.208 g, 1 mmol) and ammonium
acetate (1.27 g, 16.5 mmol) were dissolved in hot glacial acetic acid
(10 ml). While the mixture was stirred, a solution of (S)-1-(3,4-
dihydroxybutyl)-1H-indole-3-carbaldehyde (4, 0.233 g, 1 mmol)
in (15 ml) of glacial acetic acid was added dropwise. The mixture
was heated at 90 °C for 72 h and was then poured into water
(200 ml). The mixture was neutralized with aq ammonia to pH 7
and cooled to room temperature. The precipitate was filtered off
and washed with large portions of water. The residue was purified
by silica gel column chromatography [SiO2, EtOAc/Cyclohex-
ane = 1:99, (v/v)] to give 6 (0.39 g, 76%) as a faint yellow solid; Rf
0.7 (1:99 EtOAc/Cyclohexane); mp 88–90 °C. 1H NMR (DMSO) d:
2.00 (s, 3H, CH3), 2.01 (s, 3H, CH3), 2.22 (m, 2H, CH2CH2N), 4.11
(dd, J = 12.0 and 5.8 Hz, 1H, CHHN), 4.26 (dd, J = 12.0 and 3.2 Hz,
1H, CHHN), 4.42 (t, J = 7.1 Hz, 2H, CH2OCOCH3), 5.07 (m, 1H, CHO-
COCH3), 7.22-7.38 (m, 2H, ArH), 7.62 (m, 3H, ArH), 7.73 (t,
J = 7.1 Hz, 1H, ArH), 7.77 (t, J = 7.1 Hz, 1H, ArH), 8.23 (s, 1H, indole),
8.45 (d, J = 8.0 Hz, 1H, ArH), 8.68 (d, J = 7.9 Hz, 1H, ArH), 8.73 (dd,
J = 6.0 and 2.8 Hz, 1H, ArH), 8.85 (d, J = 8.3 Hz, 1H, ArH), 8.88 (d,
J = 8.4 Hz, 1H, ArH), 13.12 (s, 1H, NH). 13C NMR (DMSO) d: 20.50
(CH3), 20.72 (CH3), 30.71 (CH2CH2N), 42.41 (CH2CH2N), 64.23
(CH2OH), 69.22 (CHOH), 106.64, 110.21, 120.46, 121.52, 121.86,
121.99, 122.30, 122.39, 123.63, 124.07, 124.68, 125.70, 126.09,
126.94, 126.99, 127.17, 127.80, 136.25, 136.85, 146.87 (Ar),
170.05 (C@O), 170.16 (C@O). IR (cmꢀ1) 3419 (b, NH); 2929 (s,
7. Experimental
7.1. General
NMR spectra were recorded on a Bruker 400 MHz spectrometer
at 400 MHz for 1H, 101 MHz for 13C, and 162 MHz for 31P with TMS
as an internal standard for 1H NMR. Chemical shifts are reported in
ppm, relative to solvents peaks (CDCl3: 7.26 ppm for 1H and
77.0 ppm for 13C; DMSO-d6: 2.50 ppm for 1H and 39.5 ppm for
13C; 85% aq H3PO4 as an external standard: 0.00 ppm for 31P
NMR). Electrospray ionization high resolution mass spectra (ESI-
HRMS) were performed on PE SCIEX API Q-Star Pulsar Mass Spec-
trometer. For accurate ion mass determinations, the (MH+) or
(MNa+) ion was peak matched by calibration with NaI. Melting
points were determined on a Büchi melting point apparatus and
are not corrected. An IR spectrum was run on Perkin Elmer 1720
Fourier Transform Infrared Spectrophotometer. DCM was always
used freshly distilled and solvents used for column chromatogra-
phy of final phosphoroamidite were distilled prior to use, others
are used as received. Reagents were used as purchased. Silica gel
(0.040–0.063 mm) used for column chromatography and analytical
silica gel TLC plates 60 F254 precoated aluminium plates were pur-
chased from Merck. TLC spots of DMT containing compounds was
visualized as orange or dark spots when treated with 5% ethanolic
H2SO4 solution.
CH3); 1740 (s, C@O); 1619 (s, C@N). HRMS (ESI) m/z Calcd for
+
C
31H28N3O4 (MH+) 506.2075. Found: 506.2061.
7.1.1. (S)-1-(3,4-Dihydroxybutyl)-1H-indole-3-carbaldehyde (4)
To a stirred mixture of (S)-2,2-dimethyl-1,3-dioxolan-4-yl etha-
nol 1 (1.65 g, 11.3 mmol) and methanesulfonyl chloride (MsCl)
(1.81 g, 15.8 mmol) in dichloromethane (CH2Cl2) (20 ml) was
added dropwise triethylamine (Et3N) (1.60 g, 15.8 mmol) at ice-
bath temperature. After 2 h, the reaction mixture was partitioned
between CH2Cl2 and water. The organic layer was washed with
brine, dried (MgSO4), and concentrated in vacuo to give the meth-
anesulfonate 2 (2.5 g) as a colorless oil. This material was used
immediately without further purification.
7.1.3. (S)-4-(3-(1H-Phenanthro[9,10-d]imidazol-2-yl)-1H-indol-
1-yl)butane-1,2-diol (7)
Diacetylated compound 6 (168 mg, 0.33 mmol) was dissolved in
saturated NH3/MeOH (20 ml), and the resulting solution was stir-
red at room temperature for 72 h. The solvent was removed under
reduced pressure. The residue was suspended in dichloromethane
and filtration afforded 110 mg (78%) of 7 as a yellow solid in a state
of purity; Rf 0.2 (1:15 CH3OH/DCM); mp: 267–268 °C. 1H NMR
(DMSO) d: 1.74-2.19 (m, 2H, CH2CH2N), 3.31 (dt, J = 11.0 and
5.7 Hz, 1H, CHHOH), 3.41 (m, 2H, CHHOH+ CHOH), 4.50-4.39 (m,
2H, CH2N), 4.69 (t, J = 5.5 Hz, 1H, CH2OH), 4.90 (d, J = 5.1 Hz, 1H,
CHOH), 7.36–7.28 (m, 2H, ArH), 7.67-7.59 (m, 3H, ArH), 7.76 (t,
J = 7.5 Hz, 2H, ArH), 8.27 (s, 1H, 2-H indole), 8.45 (d, J = 7.9 Hz,
1H, ArH), 8.69 (d, J = 7.7 Hz, 1H, ArH), 8.73 (dt, J = 6.6 and 2.7 Hz,
1H, ArH), 8.84 (d, J = 8.4 Hz, 1H, ArH), 8.87 (d, J = 8.4 Hz, 1H,
ArH), 13.18 (s, 1H, NH). 13C NMR (DMSO) d: 33.91 (CH2CH2N),
42.87 (CH2CH2N), 65.78 (CH2OH), 68.47 (CHOH), 106.15, 110.29,
120.36, 121.48, 121.91, 122.30, 123.61, 124.04, 124.69, 125.67,
126.05, 126.95, 127.15, 128.20, 136.30, 136.83, 147.05 (Ar). HRMS
(ESI) m/z Calcd for C27H24N3O2+ (MH+) 422.1863. Found: 422.1855.
Anal. Calcd for C27H23N3O2ꢁ0.5 H2O: C, 75.33; H, 5.62; N, 9.76.
Found: C, 75.51; H, 5.35; N, 9.62.
To a suspension of NaH (60% in mineral oil, 506 mg, 14 mmol)
in DMF (10 ml) and Et3N (3 ml) a solution of indole-3-carboxalde-
hyde 3 (1.00 g, 7 mmol) in dry DMF (10 ml) was added dropwise
under argon. The reaction mixture was stirred for 1 h before a
solution of the above prepared methanesulfonate (2) (2.35 g,
10.5 mmol) in DMF (10 ml) was added. The resulting mixture
was stirred for 72 h at 55-60 °C. The reaction mixture was cooled
to room temperature. 1 M HCl was then added and the mixture
was stirred for 2 h. The mixture was diluted with EtOAc and ex-
tracted with satd aq Na2CO3 and satd aq NH4Cl. The organic layer
was dried (MgSO4), and concentrated in vacuo. The residue was
purified by column chromatography [SiO2, DCM/EtOAc = 10:90
(v/v)] to give 4 (0.79 g, 49%) as a yellow solid; Rf 0.1 (1:9
EtOAc/Cyclohexane); mp 98–100 °C. 1H NMR (DMSO) d: 1.74
(m, 1H, CHHCH2N), 2.04 (m, 1H, CHHCH2N), 3.18–3.30 (m, 1H,
CHHOH), 3.29–3.49 (m, 2H, CHOH, CHHOH), 4.27-4.48 (m, 2H,
CH2N), 4.57 (t, J = 5.5 Hz, 1H, CH2OH), 4.81 (d, J = 4.9 Hz, 1H,
CHOH), 7.26 (t, J = 7.4 Hz, 1H, indole), 7.32 (t, J = 7.6 Hz, 1H, in-
dole), 7.63 (d, J = 8.1 Hz, 1H, indole), 8.12 (d, J = 7.8 Hz, 1H, in-
7.1.4. (S)-4-(3-(1H-Phenanthro[9,10-d]imidazol-2-yl)-1H-indol-
1-yl)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)butan-2-ol (8)
(S)-4-(3-(1H-Phenanthro[9,10-d]imidazol-2-yl)-1H-indol-1-yl)-
butane-1,2-diol (7, 150 mg, 0.36 mmol) was coevaporated with
anhydrous pyridine (2ꢂ20 ml) and dissolved in anhydrous pyri-
dine (20 ml) and Et3N (2.5 ml). 4,40-Dimethoxytrityl chloride
dole), 8.30 (s, 1H, 2-H indole), 9.92 (s, 1H, CHO).13
C NMR

M. I. Fatthalla et al. / Bioorg. Med. Chem. 20 (2012) 207–214
213
(160 mg, 1.3 mmol) was added under a nitrogen atmosphere, and
the reaction mixture was stirred at room temperature for 72 h.
The reaction was quenched by addition of MeOH (2 ml) followed
by addition of DCM/Et3N (1%) and washed twice with saturated
aq NaHCO3. The organic phases were washed with brine, dried
(MgSO4), filtered and evaporated under diminished pressure. The
residue was coevaporated twice with toluene/EtOH 15 ml, (1:1,
v/v). The residue was purified by column chromatography [SiO2,
NEt3/CH3OH/DCM = 1:0.5:98.5, (v/v/v)] to afford the DMT pro-
tected diol 8 as a yellow solid. (220 mg, 85%); Rf 0.75 (1:15
CH3OH/DCM); mp: 165-166 °C. 1H NMR (CDCl3) d: 1.84-2.12 (m,
2H, CH2CH2N), 2.97–3.07 (m, 2H, CH2ODMT), 3.70 (s, 6H, 2ꢂOCH3),
3.75 (m, 1H, CHOH), 4.27–4.53 (m, 2H, CH2N), 4.64 (s, 1H, CHOH),
6.72 (dd, J = 8.9 and 1.2 Hz, 4H, DMT), 7.09–7.27 (m, 8H, ArH), 7.28-
7.31 (m, 1H, phenyl), 7.32-7.40 (m, 3H, ArH), 7.52 (s, 2H, ArH), 7.56
(s, 2H, ArH), 8.65 (d, J = 8.3 Hz, 2H, ArH), 8.84–8.74 (m, 2H, ArH),
8.87 (s, 2H, ArH), 13.47 (s, 1H, NH). 13C NMR (CDCl3) d: 33.37
(CH2CH2N), 42.85 (CH2CH2N), 55.19 (2ꢂOCH3), 66.52 (CHOH),
67.72 (CH2ODMT), 85.75 (OCPh3), 106.18, 109.72, 113.01, 120.62,
122.24, 122.30, 123.31, 124.58, 126.47, 126.65, 126.92, 127.62,
127.71, 128.25, 130.07, 130.09, 130.79, 136.09, 136.34, 144.89,
tem (Buffer A [0.05 M triethylammonium acetate (pH 7.4)] and
Buffer B (75% MeCN/25% Buffer A)). Flow 2.5 ml/min. Gradients:
2 min 100% A, linear gradient to 70% B in 38 min, linear gradient
to 100% B in 7 min and then 100% A in 10 min). 80% Acetic acid
were added to ODNs over 20 min, afterwards 100
tered water, aqueous AcONa (3 M, 15 L) and aqueous Sodium
perchlorate (5 M, 15 L) were then added and the ONs were pre-
lL double fil-
l
l
cipitated from pure acetone. All modified ODNs were confirmed
by MALDI-TOF analysis on a Ultraflex II TOF/TOF system from
Bruker (a MALDI-LIFT system) with HPA-matrix (10 mg 3-
hydroxypicolinic acid, in 50 mM ammoniumcitrate/70% acetoni-
tril) matrix. ODN Found m/z (Calculated m/z): ON2 4609.0
(4603.8), ON5 4609.9 (4616.8), ON6 4916.9 (4921.0) and ON7
4925.6 (4921.0). The purity of the final TFOs was found to be
over 95%, checked by ion-exchange chromatography using La-
Chrom system from Merck Hitachi on Dionex DNAPac Pa-100,
4 ꢂ 250 mm Analytical column. Melting temperature measure-
ments were performed on a Perkin–Elmer UV–Vis spectrometer
Lambda 35 fitted with a PTP-6 temperature programmer. The tri-
plexes were formed by first mixing the two strands of the Wat-
son–Crick duplex, at a concentration of 1.0 or 1.5 lM according
to the method mentioned above in the Thermal stability studies
147.89, 158.33 (Ar). HRMS (ESI) m/z Calcd for C48H42N3O4 (MH+)
+
724.317. Found: 724.3193.
part, followed by addition of the third (TFO) strand at a concen-
tration of 1.5 lM in a buffer consisting of sodium cacodylate
7.1.5. (S)-4-(3-(1H-Phenanthro[9,10-d]imidazol-2-yl)-1H-indol-
1-yl)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)butan-2-yl 2-
cyanoethyl diisopropylphosphoramidite (9)
(20 mM), NaCl (100 mM), and MgCl2 (10 mM) at pH 5.0, pH 6.0
or 7.2. TWJs were formed by mixing the two strands each at a
concentration of 1.0 lM in sodium phosphate buffer (15 mM)
DMT-protected compound 8 (0.22 g, 0.3 mmol) was dissolved
under an argon atmosphere in anhydrous CH2Cl2 (15 ml). N,N-
containing NaCl (100 mM) and EDTA (0.1 mM) at pH 7.0. The
solutions were heated to 80 °C then cooled down to 10 °C and
were then kept at this temperature for 30 min. The melting tem-
perature (Tm, °C) was determined as the maximum of the first
derivative plots of the melting curves obtained by absorbance
at 260 nm against increasing temperature (1.0 °C /min). If
needed experiments were also done at 373 nm.
´
Diisopropylammonium tetrazolide (115 mg, 0.68 mmol) was
´ ´
added, followed by dropwise addition of 2-cyanoethyl N,N,N,N-tet-
raisopropylphosphordiamidite (410 mg, 1.35 mmol) under exter-
nal cooling with an ice-water bath. The reaction mixture was
stirred at room temperature for 48 h before quenching with H2O
(10–20 ml). The layers were separated and the organic phase was
washed with H2O (10–20 ml). The combined water layers were ex-
tracted with CH2Cl2 (25 ml). The combined organic phase was
dried (Na2SO4) and filtered, and the solvent was evaporated in va-
cuo. The residue was purified by column chromatography [SiO2,
NEt3/EtOAc/cyclohexane = 1: 20:79, (v/v/v)] to afford the final
product 9 as a yellow solid (140 mg, 50%); Rf 0.73 (1:4 EtOAc/cyclo-
hexane), which was used in DNA synthesis after drying under
diminished pressure. 13C NMR (CDCl3) d: 21.02 (CH2CN), 22.67
(CH3), 22.82 (CH3), 23.44 (CH3), 24.62 (CH3), 33.67 (CH2CH2N),
43.07 (CH2CH2N), 45.58 (NCH(CH3)2), 45.63 (NCH(CH3)2), 55.18
(2ꢂOCH3), 60.34 (OCH2CH2CN), 68.18 (CHOP), 69.25 (CH2ODMT),
86.07 (OCPh3), 99.96, 109.58, 113.11, 120.79, 122.46, 126.74,
127.80, 128.07, 129.11, 130.02, 135.92, 144.79, 158.45 (Ar). 31P
NMR (CDCl3) d: 148.06, 148.69 in a 1:3 ratio. HRMS (ESI) m/z Calcd
for C57H58N5O5PNa+ (MNa+) 946.4068. Found: 946.4062.
7.3. Fluorescence measurements
The fluorescence measurements were measured on a Perkin–El-
mer LS-55 luminescence spectrometer fitted with a julabo F25
temperature controller set at 10 °C in the buffer 20 mM sodium
cacodylate, 100 mM NaCl, and 10 mM MgCl2 at pH 6.0 for triplex
formation using the same solution used for Tm measurements, set-
ting excitation and emission slits to 4 and 2.5 nm respectively and
upon excitation at 350 nm. For TWJs formation, the fluorescence
instrument was set at 10 °C in the buffer 15 mM sodium phos-
phate, 100 mM NaCl and 0.1 mM EDTA at pH 7.0, upon excitation
at 330 nm, using a 1.0 lM concentration of both hairpin strands
and modified oligonucleotide ON5 and setting excitation and emis-
sion slits were set to 4 and 0.0 nm respectively. The 0.0 nm slit is
not completely closed and allowed sufficient light to pass for the
measurement.
7.2. Oligonucleotide synthesis, purification, and melting
temperature determination
7.4. Molecular modeling
DMT-off oligodeoxynucleotides were carried out at 0.2 lmol
scales on 500 Å CPG supports with an ExpediteTM Nucleic Acid
Synthesis System Model 8909 from Applied Biosystems with
1H-tetrazole as an activator for coupling reaction. The amidite
9 was dissolved in dry DCM and inserted into the growing oligo-
nucleotides chain using an extended coupling time (15 min).
DMT-off oligonucleotides bound to CPG supports were treated
with aqueous ammonia (32%, 1 ml) at room temperature for
20 min and then at 55 °C overnight. Purification of DMT-off
ONs was accomplished by reversed-phase semipreparative HPLC
Molecular modeling was performed with Macro Model v9.1
from Schrödinger. All calculations were conducted with AMBER*
force field and the GB/SA water model. The dynamic simulations
were preformed with stochastic dynamics, a SHAKE algorithm to
constrain bonds to hydrogen, time step of 1.5 fs and simulation
temperature of 300 K. Simulation for 0.5 ns with an equilibration
time of 150 ps generated 250 structures, which all were minimized
using the PRCG method with convergence threshold of 0.05 kJ/mol.
The minimized structures were examined with Xcluster from
Schrödinger, and representative low-energy structures were se-
lected. The starting structures were generated with Insight II
on a Waters Xterra MS C18 5
l
m, 7.8ꢂ150 mm column with a
Waters Xterra MS C18 5
lm, 7.8ꢂ10 mm Chromatography Sys-

214
M. I. Fatthalla et al. / Bioorg. Med. Chem. 20 (2012) 207–214
18. Orr-Weaver, T. L.; Szostak, J. W.; Rothstein, R. J. Proc. Natl. Acad. Sci. U.S.A. 1981,
78, 6354.
v97.2 from MSI, followed by incorporation of the modified
nucleotide.
19. Guthrie, C.; Patterson, B. Annu. ReV. Genet. 1988, 22, 387.
20. (a) Wimberly, B. T.; Brodersen, D. E.; Clemons, W. M.; Morgan-Warren, R. J.;
Carter, A. P.; Vonrhein, C.; Hartsch, T.; Ramakrishnan, V. Nature 2000, 407, 327;
(b) Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A. Science 2000, 289,
905.
Acknowledgments
This work was supported by the Nucleic Acid Center, which is
funded by The Danish National Research Foundation for studies
on nucleic acid chemical biology; also a part of the funding was
supported by the Cultural Affairs & Missions Sector, Ministry of
Higher Education, Egypt.
21. Singleton, M. R.; Scaife, S.; Wigley, D. E. Cell 2001, 107, 79.
22. Hutchins, C. J.; Rathjen, P. D.; Forster, A. C.; Symons, R. H. Nucleic Acids Res.
1986, 14, 3627.
23. Pearson, C. E.; Tam, M.; Wang, Y. H.; Montgomery, S. E.; Dar, A. C.; Cleary, J. D.;
Nichol, K. Nucleic Acids Res. 2002, 30, 4534.
24. La Bean, H. T.; Yan, H.; Kopatsch, J.; Liu, F. R.; Winfree, E.; Rief, J. H.; Seeman, N.
C. J. Am. Chem. Soc. 2000, 112, 1848.
25. Muhuri, S.; Mimura, K.; Miyoshi, D.; Sugimoto, N. J. Am. Chem. Soc. 2009, 131,
9268.
Supplementary data
26. Leitner, D.; Schröder, W.; Weisz, K. Biochemistry 2000, 39, 5886.
27. Fox, K. R. Curr. Med. Chem. 2000, 7, 17.
28. Brunet, E.; Corgnali, M.; Perrouault, L.; Roig, V.; Asseline, U.; Sørensen, M. D.;
Babu, B. R.; Wengel, J.; Giovannangeli, C. Nucleic Acids Res. 2005, 33, 4223.
29. Olsen, A. G.; Dahl, O.; Nielsen, P. E. Nucleosides, Nucleotides Nucleic Acids 2003,
22, 1331.
30. Christensen, U. B.; Pedersen, E. B. Nucleic Acids Res. 2002, 30, 4918.
31. Natsume, T.; Ishikawa, Y.; Dedachi, K.; Tsukamoto, T.; Kurita, N. Chem. Phys.
Lett. 2007, 434(1–3), 133.
32. (a) Arya, D. P.; Coffee, R. L., Jr.; Charles, I. J. Am. Chem. Soc. 2001, 123, 11093; (b)
Arya, D. P. Acc. Chem. Res. 2011, 44, 134; (c) Shaw, N. N.; Arya, D. P. Biochimie
2008, 90, 1026; (d) Xue, L.; Xi, H.; Kumar, S.; Gray, D.; Davis, E.; Hamilton, P.;
Skriba, M.; Arya, D. P. Biochemistry 2010, 49, 5540.
Supplementary data (thermal denaturation first derivative plots
of matched and mismatched triplex melting (ON1/D1-4 and ON2/
D1-4) in addition to DNA/RNA TWJs melting (ON5/ON11-15) and
ON8/ON11-15); DNA TWJ heating/annealing profile for ON5/
ON11; UV absorption spectrum of M diol (Supplementary data,
Fig. S4) and 1H NMR of prepared compounds) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2011.11.013.
33. Johnson, H. A.; Thomas, N. R. Bioorg. Med. Chem. Lett. 2002, 12, 237.
34. Chen, C.; Song, G.; Yang, X.; Ren, J.; Qu, X. Biochimie 2010, 92, 1416.
35. (a) Filichev, V. V.; Pedersen, E. B. J. Am. Chem. Soc. 2005, 127, 14849; (b) Pandey,
P. C.; Weetall, H. Appl. Biochem. Biotechnol. 1995, 55, 87.
36. Filichev, V. V.; Gaber, H.; Olsen, T. R.; Jørgensen, P. T.; Jessen, C. H.; Pedersen, E.
B. Eur. J. Org. Chem. 2006, 17, 3960.
37. Osman, A. M. A.; Jørgensen, P. T.; Bomholt, N.; Pedersen, E. B. Bioorg. Med. Chem.
2008, 16, 9937.
38. Barbaric, J.; Wanninger-Weiß, C.; Wagenknecht, H. Eur. J. Org. Chem. 2009, 3,
364.
39. Bähn, S.; Imm, S.; Mevius, K.; Neubert, L.; Tillack, A.; Williams, J. M. J.; Beller, M.
Chem. Eur. J. 2010, 16, 3590.
40. Kim, B. Y.; Ahn, J. B.; Lee, H. W.; Moon, K. S.; Sim, T. B.; Shin, J. S.; Ahn, S. K.;
Hong, C. I. Chem. Pharm. Bull. 2003, 51, 276.
41. Zahran, M. A.; Ibrahim, A. M. J. Chem. Sci. 2009, 121, 455.
42. (a) Billes, F.; Podea, P. V.; Ziegler, I. M.; Tosßa, M.; Mikosch, H.; Irimie, D.
Spectrochim. Acta, Part A 2009, 74, 1031; (b) Giraud, F.; Loge, C.; Pagniez, F.;
Crepin, D.; Barres, S.; Picot, C.; Le Pape, P.; Le Borgne, M. J. Enzyme Inhibition
Med. Chem. 2009; 24, 1067.
43. Terasaka, T.; Kinoshita, T.; Kuno, M.; Seki, N.; Tanaka, K.; Nakanishi, I. J. Med.
Chem. 2004, 47, 3730.
44. Krebs, F. C.; Spanggaard, H. J. Org. Chem. 2002, 67, 7185.
45. Mansurova, M.; Koay, M. S.; Gärtner, W. Eur. J. Org. Chem. 2008, 32, 5401.
46. Gillaizeau, I.; Lagoja, I. M.; Nolan, S. P.; Aucagne, V.; Rozenski, J.; Herdewijn, P.;
Agrofoglio, L. A. Eur. J. Org. Chem. 2003, 4, 666.
47. Rougbe, M.; Faucon, B.; Mergny, J. L.; Barcelo, F.; Giovannangeli, C.; Garestier,
T.; Hèléne, C. Biochemistry 1992, 31, 9269.
48. Bomholt, N.; Osman, A. M. A.; Pedersen, E. B. Org. Biomol. Chem. 2008, 6, 3714.
References and notes
1. Felsenfeld, G.; Rich, A. Biochim. Biophys. Acta 1957, 26, 457.
2. Thenmalarchelvi, R.; Yathindra, N. Nucleic Acids Res. 2005, 33, 43.
3. Goni, J. R.; De La Cruz, X.; Orozco, M. Nucleic Acids Res. 2004, 32, 354.
4. Sargent, R. G.; Kim, K. Dieter C.; Gruenert, D. C. Oligonucleotides 2011, 21, 55.
5. Htun, H.; Dahlberg, J. E. Science 1989, 243, 1571.
6. Jain, A. K.; Bhattacharya, S. Bioconjugate Chem. 2010, 21, 1389.
7. Duca, M.; Vekhoff, P.; Oussedik, K.; Halby, L.; Arimondo, P. B. Nucleic Acids Res.
2008, 36, 5123.
8. Arimondo, P. B.; Boutorine, A.; Baldeyrou, B.; Bailly, C.; Kuwahara, M.; Hecht, S.
M.; Sun, J. S.; Garestier, T.; Helene, C. J. Biol. Chem. 2002, 277, 3132.
9. Panakanti, R.; Pratap, A.; Yang, N.; Jackson, J. S.; Mahato, R. I. Biochem.
Pharmacol. 2010, 80, 1718.
10. Sedelnikova, O. A.; Karamychev, V. N.; Panyutin, I. G.; Neumann, R. D. Antisense
Nucleic Acid Drug Dev. 2002, 12, 43.
11. Besch, R.; Marschall, C.; Schuh, T.; Giovannangeli, C.; Kammerbauer, C.; Degitz,
K. J. InVest. Dermatol. 2004, 122, 1114.
12. Hahn, W. C.; Counter, C. M.; Lundberg, A. S.; Roderick, L.; Beijersbergen, R. L.;
Mary, W.; Brooks, M. W.; Robert, A.; Weinberg, R. A. Nature 1999, 400, 464.
13. Samadashwily, G. M.; Mirkin, S. M. Gene 1994, 149, 127.
14. Francois, J. C.; Saison-Behmoaras, T.; Thoung, N. T.; Helene, C. Biochemistry
1989, 28, 9617.
15. Maher, L. J.; Wold, B.; Dervan, P. B. Science 1989, 245, 725.
16. Roulon, T.; Coulaud, D.; Delain, E.; Le Cam, E.; Hélène, C.; Escudé, C. Nucleic
Acids Res. 2002, 30, e-12.
17. Prakash, G.; Kool, E. J. Am. Chem. Soc. 1992, 114, 523.