Using an aryl phenanthroimidazole moiety as a conjugated flexible intercalator to improve the hybridization efficiency of a triplex-forming oligonucleotide

Article abstract of DOI:10.1016/j.bmc.2008.10.037

When inserting 2-phenyl or 2-naphth-1-yl-phenanthroimidazole intercalators (X and Y, respectively) as bulges into triplex-forming oligonucleotides, both intercalators show extraordinary high thermal stability of the corresponding Hoogsteen-type triplexes and Hoogsteen-type parallel duplexes with high discrimination to Hoogsteen mismatches. Molecular modeling shows that the phenyl or the naphthyl ring stacks with the nucleobases in the TFO, while the phenanthroimidazol moiety stacks with the base pairs of the dsDNA. DNA-strands containing the intercalator X show higher thermal triplex stability than DNA-strands containing the intercalator Y. The difference can be explained by a lower degree of planarity of the intercalator in the case of naphthyl. It was also observed that triplex stability was considerably reduced when the intercalators X or Y was replaced by 2-(naphthlen-1-yl)imidazole. This confirms intercalation as the important factor for triplex stabilization and it rules out an alternative complexation of protonated imidazole with two phosphate groups. The intercalating nucleic acid monomers X and Y were obtained via a condensation reaction of 9,10-phenanthrenequinone (4) with (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)benzaldehyde (3a) or (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)-1-naphthaldehyde (3b), respectively, in the presence of acetic acid and ammonium acetate. The required monomers for DNA synthesis using amidite chemistry were obtained by standard deprotection of the hydroxy groups followed by 4,4′-dimethoxytritylation and phosphitylation.

Full text of DOI:10.1016/j.bmc.2008.10.037

Bioorganic & Medicinal Chemistry 16 (2008) 9937–9947
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Using an aryl phenanthroimidazole moiety as a conjugated flexible
intercalator to improve the hybridization efficiency of a triplex-forming
oligonucleotide
*
Amany M. A. Osman , Per T. Jørgensen, Niels Bomholt, Erik B. Pedersen
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
When inserting 2-phenyl or 2-naphth-1-yl-phenanthroimidazole intercalators (X and Y, respectively) as
bulges into triplex-forming oligonucleotides, both intercalators show extraordinary high thermal stabil-
ity of the corresponding Hoogsteen-type triplexes and Hoogsteen-type parallel duplexes with high dis-
crimination to Hoogsteen mismatches. Molecular modeling shows that the phenyl or the naphthyl ring
stacks with the nucleobases in the TFO, while the phenanthroimidazol moiety stacks with the base pairs
of the dsDNA. DNA-strands containing the intercalator X show higher thermal triplex stability than DNA-
strands containing the intercalator Y. The difference can be explained by a lower degree of planarity of
the intercalator in the case of naphthyl. It was also observed that triplex stability was considerably
reduced when the intercalators X or Y was replaced by 2-(naphthlen-1-yl)imidazole. This confirms
intercalation as the important factor for triplex stabilization and it rules out an alternative complexation
of protonated imidazole with two phosphate groups. The intercalating nucleic acid monomers X and Y
Received 19 May 2008
Revised 3 October 2008
Accepted 12 October 2008
Available online 17 October 2008
Keywords:
Intercalating nucleic acids
Phenanthroimidazole
DNA Triplex stability
Molecular modeling
were obtained via
a condensation reaction of 9,10-phenanthrenequinone (4) with (S)-4-(2-(2,2-
dimethyl-1,3-dioxolan-4-yl)ethoxy)benzaldehyde (3a) or (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)
ethoxy)-1-naphthaldehyde (3b), respectively, in the presence of acetic acid and ammonium acetate.
The required monomers for DNA synthesis using amidite chemistry were obtained by standard deprotec-
tion of the hydroxy groups followed by 4,40-dimethoxytritylation and phosphitylation.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ylethynyl) benzyl]- glycerol (W, TINA, Fig. 1) was inserted as a
bulge in the middle of a TFO.28
The ability of triplex-forming oligonucleotides (TFOs) to inter-
act specifically with polypurine/polypyrimidine double-stranded
DNA forming triplexes has shown them as candidates for regula-
tion of transcription of genomic DNA in the so-called antigene
strategy.1–7 Moreover, TFOs induce gene recombination and repair-
ing genetic defects in mammalian cells.8–10 However, in many
cases triplexes are thermodynamically less stable than correspond-
ing duplexes. For this reason, an enormous number of oligodeoxy-
nucleotides (ODN) have been developed, either by modifying the
nucleobase,11–13 the sugar part,14–19 or the phosphate back-
bone20–27 to improve triplex stabilization. The triplex stabilization
can also be achieved by insertion of different intercalating agents.
Recently, we observed the extraordinary stable Hoogsteen-type
triplexes and duplexes, when the intercalator (R)-1-O-[4-(1-pyren-
The phenyl group of TINA is supposed to stack with the nucleo-
bases in the TFO, while the pyrene moiety is expected to intercalate
with the nucleobases of the duplex. In our search for triple-helix spe-
cific binding compounds that would function under physiological
conditions; we have used the conjugated intercalator W to avoid
self-association or quadruplex formation of G-rich oligonucleotides.
Contrary to wild-type oligonucleotides, the corresponding oligonu-
cleotides with W insertions were able to form stable triplexes in
the presence of a physiologic potassium ion concentration.29 How-
ever, polycyclic aromatic hydrocarbons such as phenanthrene and
pyrene are carcinogenic environmental pollutants.30 Therefore, the
1H-phenanthro[9,10-d]imidazol-2-yl group is worth considering to
substitute thepyrenylconjugatedsystem of W. It hasmanydesirable
properties such as good stability, ease of introduction into molecular
system containing an aromatic aldehyde, use as a chromophore with
high extinction coefficient, readily tuneable absorption wavelength,
and fluorophoric properties. More recently, 2-(phenyl or heterocy-
clic)-1H-phenanthro[9,10-d]imidazole has been reported as the
inhibitor of microsomal prostaglandin E synthase-1 (mPGES-1) en-
zyme and it has been used for the treatment of various diseases, such
* Corresponding author. Tel.: +45 65502555; fax: +45 66158780.
E-mail address: ebp@ifk.sdu.dk (E.B. Pedersen).
On leave from Chemistry Department, Faculty of Science, Menoufia University,
Shebin El-Koam, Egypt.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.037

9938
A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
NH
N
N
NH
NH
N
O
O
O
DNA
O
DNA
O
O
DNA
O
O
O
O
O
O
DNA
O
P
P
O
P
O
DNA
DNA
DNA
O
O
O
O
P
DNA
O
Z
X
Y
W
Figure 1. The synthesized intercalators X, Y, and Z with the reference intercalator W (TINA).
as osteoarthritis and rheumatoid arthritis.31 The introduction of a
fused imidazol ringwith a larger aromatic system was believed to re-
sult in a higher affinity for dsDNA. In the present work, we describe
the synthesis of two intercalating nucleic acids X and Y to enhance
the stability of the TFO using a short flexible linker. The linker of
the intercalators X, Y, and Z have the same number of atoms as the
intercalator W, but for the ease of synthesis an oxygen atom and a
carbon atom have been interchanged so that the oxygen is directly
attached to the phenyl or the naphthyl ring. Thermal denaturation
studies with the triplex-forming oligonucleotide proved that the
intercalator X induced an enhanced triplex stability compared to
wild type. From molecular modeling, it is found that intercalation
is not necessarily the only possible explanation for triplex stabiliza-
tion. The modeling showed also a possible active participation by a
protonated imidazole ring which can have ionic interactions and
form hydrogen bondings to two phosphate groups in two different
backbones. However, the alternative mode of triplex stabilization
was ruled out by synthesizing the truncated intercalator Z which
gave less stable parallel triplexes, when inserted as a bulge.
reaction with 4,40-dimethoxytrityl chloride (DMT-Cl) in anhydrous
pyridine at room temperature under a N2 atmosphere. Silica gel
purification afforded the DMT-protected compounds 7a,b in 79%
and 56% yield, respectively. The secondary hydroxy group of these
compounds was phosphitylated overnight with 2-cyanoethyl
N,N,N0,N0-tetraisopropyl phosphorodiamidite in the presence of
diisopropyl ammonium tetrazolide as activator in anhydrous CH2Cl2
to afford 8a,b in 86% and 81% yield, respectively (Scheme 1).
It was believed that the corresponding imidazolyl amidite
derivative 13 without the phenanthrene ring system could be eas-
ily obtained from the corresponding monomer 10 (Scheme 2). In
order to synthesize the monomer 10, compound 3b was deprotec-
ted with 80% aqueous acetic acid to give (S)-4-(3,4-dihydroxybut-
oxy)-1-naphthaldehyde (9) in 100% yield. This compound was
reacted in ethanol and MeCN at 0 °C with a solution of 40% glyoxal
in water and 20 M ammonium hydroxide overnight to afford (S)-4-
(4-(1H-imidazol-2-yl)naphthalene-1-yloxy)butan-1,2-diol (10) in
44% yield in analogy with the procedure of Nakumura et al.34
Unfortunately, the subsequent attempt to make the DMT-pro-
tected compound 12 failed although a variety of procedures were
investigated. Therefore, it was decided to change the synthetic
strategy. Instead, the primary hydroxyl group of compound 9
was DMT-protected to afford the compound 11 in 60% yield after
purification by column chromatography. The imadazolyl derivative
12 was then obtained in 32% yield from compound 11 using the
same reaction conditions as used for converting compound 9 into
compound 10. Finally, the amidite 13 was obtained in 81% yield
by a standard phosphitylation reaction of compound 12.
2. Chemistry
The synthetic route toward the intercalating nucleic acid mono-
mers (6a,b) is shown in Scheme 1. The key intermediates 3a,b were
synthesized from (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol
(1) by reaction with 4-hydroxybenzaldehyde (2a) or 4-hydroxy-1-
naphthaldehyde (2b) under Mitsunobu conditions32 (DEAD, THF,
and Ph3P) in high yields 81% and 92%, respectively (Scheme 1). Sub-
sequent treatment of 3a,b with phenanthrene-9,10-dione (4) and
ammonium acetate in hot glacial acetic acid according to the proce-
dure of Krebs and Spanggaard33 afforded the monomers 6a,b. When
starting from 3a the product mixture was separated by silica gel col-
umn chromatography to afford the deprotected (S)-4-(4-(1H-phen-
anthro[9,10-d]imidazol-2-yl)phenoxy)butane-1,2-diol (6a) in 72%
yield and a minor amount of the corresponding diol (5) still
protected with an isopropylidene group. Due to exchange of the
imidazole protons, a line broadening was observed in the 1H NMR
spectrum of (5). This resulted in a broad singlet for the neighboring
protons in the phenanthrene ring at C-4 and C-11. The correspond-
ing compound (S)-4-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)nap-
halen-1-yloxy)butane-1,2-diol (6b) was isolated fully deprotected
by precipitation in 80% yield without chromatographic purification.
The primary hydroxy group of compounds (6a,b) was protected by
The obtained phosphoramidites 8a,b and 13 were incorporated
into a 14-mer oligonucleotide by a standard phosphoramidite pro-
tocol on an automated DNA synthesizer. However, an extended
coupling time (10 min), in the oligonucleotide synthesis as was
used for the amidite of the natural nucleosides. All modified ODNs
were purified by reversed-phase HPLC, and confirmed by MALDI-
TOF-MS analysis as reported in Section 7. The purity of the final se-
quences was determined by ion-exchange HPLC (IE-HPLC) to be
more than 90%.
3. Thermal stability studies
The thermal stabilities of parallel triplexes and parallel du-
plexes as well as antiparallel DNA/DNA and DNA/RNA duplexes
containing the intercalators X, Y, and Z were evaluated by thermal

A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
9939
CHO
CHO
O
O
O
O
O
DEAD/THF
Ph3P
H
or
+
OH
O
R2
R1
OH
OH
1
2a
2b
3a,b
NH4OAc/AcOH
90oc
3a,b
+
+
O
O
N
NH
N
NH
R2
R1
4
O
O
O
HO
O
HO
6a,b
5
DMT-Cl
pyridine
R1
R2
a
b
H
H
benzo
N
NH
R2
R1
O
DMTO
RO
R = H
7a,b
NC(CH2)2OP(NiPr2)2
CH2Cl2, r.t.
8a,b R = P(NiPr2)O(CH2)2CN
Scheme 1.
denaturation experiments. The thermal melting studies of X and Y
were compared with the previously published data for the interca-
lator W (TINA)28a as shown in Tables 1–3. The melting tempera-
tures (Tm, °C) were determined as the first derivatives of melting
curves. Since protonation of cytosine is required to form stable
Hoogsteen bonds, thermal stability of parallel triplexes using the
synthesized oligonucleotides towards the duplex (D1)35 was as-
sessed both at pH 6.0 and pH 7.2, the ultimate goal being to find
triplex formation at physiological pH conditions. Thermal stability
of the corresponding parallel duplexes was also assessed using tar-
geting to the purine strand ON1836 (Table 1).
Stabilization of parallel triplexes was found in all cases when
compared with the wild-type ON1 at pH 6.0 and 7.2 except in case
of ON5 and ON9 with insertion of the truncated intercalator Z. At
pH 6.0, the stability of the modified sequences ON10 and ON13
with the intercalator X were also measured at a wavelength of
k = 373 nm, because of overlapping curves at k = 260 nm for triplex
and duplex melting. At pH 6 and independently of the site of inser-

9940
A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
O
O
H
O
DMTO
H
H
aq. AcOH
DMT-Cl
3b
pyridine
HO
HO
O
O
9
11
glyoxal, NH4OH, EtOH,
MeCN/H2O
glyoxal, NH4OH, EtOH,
MeCN/H2O
N
N
HO
HO
DMTO
RO
N
H
N
H
O
O
10
12 R = H
NC(CH2)2OP(NiPr2)2
CH2Cl2, r.t.
13 R = P(NiPr2)O(CH2)2CN
Scheme 2.
Table 1
Tm (°C) data for triplex and duplex melting, evaluated from UV melting curves (k = 260 nm).
Entry
TFO
Parallel triplexa
Parallel duplexb
50-GACGGGGAAAGAAAAAA (ON18)
30-CTGCCCCTTTCTTTTTT
50-GACGGGGAAAGAAAAAA (D1)
pH 5.0
pH 6.0
pH 7.2
pH 6.0
ON1
ON2
ON3
ON4
ON5
ON6
ON7
ON8
50-CCCCTTTCTTTTTT-30
50-CCCCTTWTCTTTTTT-30
50-CCCCTTXTCTTTTTT-30
50-CCCCTTYTCTTTTTT-30
50-CCCCTTZTCTTTTTT-30
50-CCCCTTTCWTTTTTT-30
50-CCCCTTTCXTTTTTT-30
50-CCCCTTTCYTTTTTT-30
50-CCCCTTTCZTTTTTT-30
50-CCCCTTTCTXTTTTT-30
50-CCCCTTTCTYTTTTT-30
50-CCCCTTWTCTWTTTTT-30
50-CCCCTTXTCTXTTTTT-30
50-CCCCTTYTCTYTTTTT-30
50-WCCCCTTTCTTTTTT-30
50-XCCCCTTTCTTTTTT-30
50-CCCCTTTCTTTTTTX-30
55.0c
59.0c
59.5
55.0
33.0
28.0
45.5
46.5
40.5
10.5
39.5c
43.5
35.5
13.5
48.5e
38.5
56.5c,e
51.5e
46.5
44.5c
46.0
43.5
<5.0
28.0
26.0
18.5
19.0
33.5c
31.5
21.5
d
d
—
—
d
—
—
—
—
—
—
—
—
21.5c
25.0
18.5
30.0c
34.5
23.0
d
d
d
d
d
ON9
—
—
d
ON10
ON11
ON12
ON13
ON14
ON15
ON16
ON17
33.5
18.5
43.0c
37.0
15.0
20.5c
20.5
20.0
31.5
19.5
38.0c
37.5
20.5
36.0c
34.0
31.5
d
d
d
d
—
—
—
d
d
d
—
a
C = 1.5
l
M of ON1–17 and 1.0
l
M of each strand of dsDNA(D1) in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 5.0, pH 6.0, and 7.2. Duplex Tm = 58.5 °C (pH
6.0) and 57.0 °C (pH 7.2).
b
C = 1.0
Data taken from Ref. 28a.
Not determined.
lM of each strand in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 6.0.
c
d
e
Third strand and duplex melting overlaid. Tm values determined at 373 nm.
tion of the intercaltor X, the triplex stabilities of ON3/D1
(Tm = 46.5 °C), ON7/D1 (Tm = 43.5 °C), and ON10/D1 (Tm = 48.5 °C)
are enormously increased compared to the unmodified triplex
ON1/D1 (Tm = 28.0 °C). The observed stabilization in the range of
more clear annealing temperature upon cooling a mixture of ON3
and D1 (Fig. 2), than the one obtained for ON2 and D1.
The importance of a large aromatic ring system as an intercala-
tor was confirmed by observing that the truncated intercalator Z
inserted as a bulge gave less stable parallel triplexes (ON5 and
ON9) when compared with the wild-type ON1 at pH 5.0 and 6.0.
As discussed later on under molecular modeling, an alternative io-
nic interaction between a protonated imidazole ring of the interca-
lator and the negative charged backbones of the triplex was found,
which encouraged us to investigate thermal stability at pH 5.0 for
the three novel intercalators. As can be seen from Table 1, a large
decrease in thermal stability upon bulge insertion of intercalator
Z (ON5) as compared to the wild-type (ON1) was detected. This
observation confirms that the stability of the triplexes with bulge
D
Tm = 15.5–20.5 °C corresponds to an excellent intercalating sys-
tem. When thermal melting using the insertions of X in ON3 and
ON7 is compared with W in ON2 and ON6 almost identical triple
stabilities are observed at pH 6.0 and 7.2 although with a small
preference of X over W in three out of four cases. The opposite
trend is observed upon double insertions when ON12/D1 is com-
pared with ON13/D1. This may reflect a stringent triplex structure
which is highly selective toward intercalators on multiple inser-
tions. Another interesting difference between the intercalators W
and X was observed in annealing experiments where X gave a

A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
9941
Table 2
Tm (°C) data for mismatched Hoogsteen parallel triplexa melting, 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
ON10
ON11
ON13
ON14
50-CCCCTTTCTTTTTT-30
28.0
45.5
46.5
40.5
48.5
38.5
51.5
46.5
<5.0
27.0b
23.0
16.5
30.5
21.0
35.5
24.0
<5.0
34.5b
29.5
21.0
33.0
22.5
37.0
33.5
<5.0
28.5b
31.5
25.5
35.5
26.0
42.0
17.5
50-CCCCTTWTCTTTTTT-30
50-CCCCTTXTCTTTTTT-30
50-CCCCTTYTCTTTTTT-30
50-CCCCTTTCTXTTTTT-30
50-CCCCTTTCTYTTTTT-30
50-CCCCTTXTCTXTTTTT-30
50-CCCCTTYTCTYTTTTT-30
a
C = 1.5
Data taken from Ref. 28a.
l
M of each oligonucleotide and 1.0
lM of each strand of dsDNA in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 6.0.
b
Table 3
Tm (°C) data for Watson–Crick antiparallel duplexes melting, evaluated from UV melting curves (k = 260 nm).
Entry
Sequences
DNAa 30-GGGGAAAGAAAAAA (ON19)
RNAb 30-r(GGGGAAAGAAAAAA) (ON20)
pH 6.0
pH 7.2
pH 7.0
52.0
ON1
ON2
ON3
ON4
ON6
ON7
ON8
ON10
ON11
ON12
ON13
ON14
ON15
ON16
ON17
50-CCCCTTTCTTTTTT-30
50-CCCCTTWTCTTTTTT-30
50-CCCCTTXTCTTTTTT-30
50-CCCCTTYTCTTTTTT-30
50-CCCCTTTCWTTTTTT-30
50-CCCCTTTCXTTTTTT-30
50-CCCCTTTCYTTTTTT-30
50-CCCCTTTCTXTTTTT-30
50-CCCCTTTCTYTTTTT-30
50-CCCCTTWTCTWTTTTT-30
50-CCCCTTXTCTXTTTTT-30
50-CCCCTTYTCTYTTTTT-30
50-WCCCCTTTCTTTTTT-30
50-XCCCCTTTCTTTTTT-30
50-CCCCTTTCTTTTTTX-30
49.5
46.5c
50.5
46.5
44.5
51.0
46.0
51.0
47.5
41.0c
49.0
38.5
53.0c
56.5
54.0
49.5
45.5c
50.5
46.0
d
-
53.0
49.5
d
d
—
—
50.5
46.0
51.0
47.5
38.0c
50.5
38.5
52.0c
56.5
54.0
51.0
49.0
53.0
49.5
d
—
49.5
42.5
d
—
59.0
55.5
a
C = 1.0
C = 1.0
Data taken from Ref. 28a.
Not reported in Ref. 28a.
l
l
M of each oligonucleotide in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 6.0 and 7.2.
M of each oligonucleotide in 140 mM NaCl, 10 mM sodium phosphate buffer, 1 mM EDTA, pH 7.0.
b
c
d
Figure 2. First derivatives plots of triplex melting (up and down) for ON3 and ON2 incorporating monomer X and W, respectively, recorded at 260 nm versus increasing
temperature (1 °C/min) in 20 mM sodium cacodylate, 100 mM NaCl, 10 mM MgCl2, pH 6.0.
insertions of X is due to intercalation and not an ionic interaction
even at low pH. It was thought an advantage to replace the ben-
zene ring in the intercalator X with the larger naphthalene ring
to obtain the intercalator Y which was believed to give better
stacking with the base pairs of the TFO. Such an effect has previ-
ously been reported for alternate strand triplexes.37 However, con-
siderably lower triplex meltings (6–15 °C at pH 6.0 and 7.2) were
observed for the Y containing oligos ON4, ON8, and ON11 than
for the X containing oligos ON3, ON7, and ON10, respectively. This
is explained under molecular modeling by sterical hindrance to
planarity when naphthalene is incorporated into the intercalator.
Attaching the intercalator X at the 50-end (ON16) gave better sta-
bilization of Hoogsteen-type triplexes and duplexes than at the
30-end (ON17).
The parallel triplexes with bulge insertion of the intercalators
W, X, and Y in the middle of the TFO were studied for their sensi-
tivity to Hoogsteen mismatches at pH 6.0 (Table 2). For mono
insertions, X was slightly better than W to discriminate neighbor-
ing Hoogsteen mismatches in ON3 (15–23.5 °C) compared to ON2
(11–18.5 °C), respectively. For X, it is approximately the same
range that is found for discrimination for a non-neighboring inser-
tion (ON10). The worst case for discrimination was actually found
when the study was extended to TFOs with double insertions of the
intercalators X and Y separated by three nucleobases. Here, the tri-
plex containing ON13/D4 gave the smallest change in
DTm =
–9.5 °C for replacement of a T/A base pair with a G/C base pair in
the duplex part of the triplex. The discriminating power of a mono
inserted intercalator should be compared with the work of Zhou et

9942
A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
al.38 who was actually aiming at stabilizing triplex forming of mis-
match. They inserted 2-methoxy-6-chloro-9-aminoacridine in the
modified TFO for the duplex and triplex measurements. Excitation
and emission slits were set to 4 and 0.0 nm, respectively, the
0.0 nm slit not being totally dark. The fluorescence spectra of the
TFO ON3 toward D1, D2, D3, and D4 were recorded at pH 6.0 and
they are shown in Figure 3A. The fluorescence intensity increased
of the fully matched triplex ON3/D1 compared to the single-
stranded ON3. However, the emission intensity of the triplex Hoogs-
teen mismatched ON3/D2 decreased slightly because of an inverted
A/T base pair in the duplex next to the intercalator compared to the
matching triplex, On the contrary, when a Hoogsteen mismatch was
due to a C/G base pair near the insertion of the intercalating X (ON3/
D3, ON3/D4), the fluorescence intensity was even lower than the
one of the single strand TFO. The fluorescence spectra of the oligo
ON3 toward ON18, ON19 in parallel and antiparallel duplexes,
respectively, are shown in Figure 3B. The emission intensity of the
antiparallel duplex ON3/ON19 is comparable to the one of the single
strand ON3 whereas the parallel duplex ON3/ON18 showed in-
creased fluorescence intensity.
middle of the TFOs as a bulge insertion and the
DTm values were
in the range of 10 °C which is a much lower discriminating power
than the ones found for our intercalators.
If the ultimate goal is to use modified TFOs as antigene oligos to
control diseases, it is also important to consider the effect of the
modification if the oligo can make stable complexes with other tar-
gets, e.g. forming a parallel duplex by Hoogsteen bonding or nor-
mal antiparallel DNA/DNA or DNA/RNA duplexes. Here, the TFOs
were targeted in a parallel duplex fashion to the oligo ON18. As
it can be seen from Table 1 considerable stabilizations (12.5–
15.5 °C at pH 6.0) are achieved for the intercalator X for mono
insertions when compared with the wild-type parallel duplex. This
is slightly lower than the stabilizations (15.5–20.5 °C at pH 6.0)
found for the corresponding triplexes. Besides it is important to
note that the triplex melting is 9–17 °C higher than the corre-
sponding parallel duplex melting.
The thermal stability studies of antiparallel Hoogsteen-type
DNA/DNA duplexes were observed at pH 6.0, pH 7.2, and the cor-
responding DNA/RNA duplex was performed at pH 7.0 (Table 3).
As shown for ON2, ON6, and ON12, destabilization has been de-
scribed for oligos including the intercalator W in the middle of
the oligo toward ON19 in antiparallel Watson–Crick-type DNA/
DNA duplexes, when compared with the wild-type duplex.28a Con-
sidering the similarity of W and X when used as conjugated bulge
intercalators in triplex studies, it was surprising to find that the
melting temperatures of both DNA/DNA and DNA/RNA duplexes
with bulging X showed nearly identical melting temperatures to
the corresponding wild-type duplexes (ON3, ON7, and ON10). This
holds even for double insertion of X (ON13). When the intercala-
tors W and X were placed at the 50-end in ON15, ON16, respec-
tively, or at the 30-end in ON17, the stabilization effect was in
5. Molecular modeling
The novel monomers X and Ys ability to stabilize the triplex via
intercalation were studied using representative low-energy struc-
tures generated with the AMBER* force field in MacroModel 9.1.
Molecular modeling was performed on truncated triplexes with
the intercalator incorporated into the middle of the triplex. As it
can be seen from Figure 4A–B, the position of the intercalators, X
and Y, are similar and in both cases are the phenanthroimidazole
moiety positioned in the Watson–Crick duplex thereby adding to
the triplex stability via p–p-interaction. In addition, the phenyl- or
naphthalene-moiety is positioned between nucleobases of the
TFO, adding to the stability as well as ensuring equal amount of
unwinding at the site of intercalation. In the case of intercalator X,
the phenyl-moiety is only slightly twisted in comparison to the
naphthalene-moiety of intercalator Y which is forced out of plane
by sterical interaction between protons on the naphthalene-moiety
and on the imidazole-moiety. The large extent of twisting between
the two aromatic moieties of Y forces the nucleobases of the TFO
to twist out of plane compared to X, thereby weakening the stacking
interactions. This conclusion supports the thermal stability mea-
surements which showed a decrease in triplex stability using inter-
calator Y in comparison with intercalator X, clearly demonstrating
the range
DTm = 3.5–7.0 °C for both DNA and RNA targeting. This
is ascribed to stacking of the aromatic system on the adjacent
nucleobases, which is known as the lid-effect.39,40
4. Fluorescence properties
The fluorescence measurements were performed for the single
strand TFO (ON3) which was found effective to form triplexes and
to discriminate Hoogsteen mismatches. The insertion of the interca-
lator X into oligonucleotides resulted in a characteristic monomeric
fluorescence spectrum, with maxima at 400 nm upon excitation at
373 nm (Fig. 3). In all cases, a 4 nm shift of monomeric fluorescence
was detected upon formation of triplexes or duplexes except in two
cases ON3/D3, ON3/D4. The spectra were recorded from 340 to
600 nm at 10 °C in the same buffer solutions use for Tm studies using
the importance of optimal p–p-stacking interactions. Twisting the
naphthalene-moiety of intercalator Y around the single bond re-
sulted in conformations with almost identical intercalating proper-
ties in the triplex and no optimal conformation could be assigned. In
addition, protonation of the imidazole ring resulted in almost iden-
tical intercalating properties of the intercalator, but the protonated
imidazole provided an alternative structure to the intercalating
a 1.0 lM concentration of each strand of the unmodified duplex and
Figure 3. Fluorescence emission spectra of ON3 incorporating monomer X upon excitation at 373 nm and pH 6.0. (A) ON3 forming parallel triplex and mismatched triplexes.
(B) ON3 forming parallel duplex and antiparallel duplex.

A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
9943
Figure 4. Representative low-energy structures triplexes intercalated by X (A) and Y (B), and the ionic interactions and hydrogen bondings between protonated Y and two
phosphate groups (C).
structure. It was found that the protonated nitrogens of the imidaz-
ole ring positionedin thegroove formedbetween the TFOand purine
target were capable of forming ionic interactions and hydrogen bon-
dings across the groove to two phosphate groups in each their back-
bone (Fig. 4C). This interaction could result in a stabilizing effect at
low pH which not resulted from intercalation. Therefore, to investi-
gate this possibility for an alternative stabilizing interaction, we syn-
thesized the intercalator Z, which lacked the phenanthrene moiety
and thereby should be unable to intercalate the triplex. The mode
of triplex stabilization could be determined by experimentally com-
paring thermal stability of Y and Z at low pH, and the structure in
Figure 4C was in that way ruled out.
TMS as an internal standard for 1H NMR, deuterated solvents CDCl3
(d 77.00 ppm), DMSO-d6 (d 39.44 ppm) for 13C NMR, and 85%
H3PO4 as an external standard for 31P NMR. MALDI mass spectra
of the synthesized compounds were recorded on a Fourier Trans-
form Ion Cyclotron Resonance Mass Spectrometer (IonSpec, Irvine,
CA). For accurate ion mass determinations, the (MH+) or (MNa+) ion
was peak matched using ions derived from the 2,5-dihydroxyben-
zoic acid matrix. Electrospray ionization mass spectra (ESI-MS)
were performed on a 4.7 T HiResESI Uitima (FT) mass spectrome-
ter. Both spectrometers are controlled by the OMEGA Data System.
Melting points were determined on a Büchi melting point appara-
tus. Silica gel (0.040–0.063 mm) used for column chromatography
and analytical silica gel TLC plates 60 F254 were purchased from
Merck. Solvents used for column chromatography were distilled
prior to use, while reagents were used as purchased. Petroleum
ether (PE): bp 60–80 °C.
6. Conclusion
Here, we have described the synthesis of two intercalating nu-
cleic acid monomers X and Y, and their incorporation into oligonu-
cleotides giving in good yield using normal oligonucleotide
synthesis procedures. Melting studies showed that the two interca-
lators have extraordinary high thermal stability of Hoogsteen-type
triplexes and duplexes with a high discrimination of mismatch
strands. DNA-strands containing intercalator X show higher ther-
mal triplex stability than DNA-strands containing intercalator Y.
Interestingly, the intercalator X (ON7) slightly increased the triplex
stability when compared to the intercalator W (TINA). In our re-
search, the linker was the same atom number of the previous stud-
ies (TINA) but differs so that the oxygen atom was attached directly
to the phenyl or naphthyl rings, respectively. The mode of interca-
lation was confirmed by the synthesis of intercalator Z which gave
less stable parallel triplexes, when inserted as a bulge which means
that the imidazol ring is too small to be an effective intercalator. In
addition, an alternative ionic interaction across the groove formed
between the TFO and purine target between protonated imidazol
ring and negative by charge backbones was ruled out based on
Tm determination at low pH.
7.1.1. General procedure for preparation of 3 in a Mitsunobu
reaction
An ice-cooled solution of diethylazodicarboxylate (DEAD, 2.5 ml,
16 mmol) in dry THF (155 ml) was treated with (S)-2-(2,2-dimethyl-
1,3-dioxolan-4-yl)ethanol (1) (1.9 ml, 13 mmol) for 25 min, and
then 4-hydroxybenzaldehyde (2a) (2.1 g, 17 mmol) or 4-hydroxy-
1-naphthaldehyde (2b) (3.0 g, 17 mmol) and triphenylphosphine
(4.2 g, 16 mmol) were added to the mixture. The mixture was stirred
in an ice-water bath for 30 min, and then allowed to warm to room
temperature overnight. The mixture was quenched with aqueous
ammonia (105 ml) and extracted with AcOEt. The organic layer
was washed with water, dried over MgSO4, and concentrated under
reduced pressure to leave an oil which was purified by silica gel col-
umn chromatography [petroleum ether/diethyl ether (1:1, v/v)] to
afford the pure products (3a,b).
7.1.2. (S)-4-(2-(2,2-Dimethyl-1,3-dioxolan-4-yl)ethoxy)benzald-
ehyde (3a)
Yield: 3.5 g (81%) as an oil; Rf 0.30 (50% petroleum ether/diethyl
ether). 1H NMR (CDCl3): d 1.38 (s, 3H, CH3), 1.44 (s, 3H, CH3), 2.08
(m, 2H, CH2CH2O), 3.67 (m, 1H, CHH), 4.12–4.22 (m, 3H, CHH and
CH2CH2O), 4.32 (m, 1H, CH), 7.01 (d, 2H, J = 8.7 Hz, aryl), 7.84 (d,
2H, J = 8.7 Hz, aryl), 9.88 (s, 1H, CHO). 13C NMR (CDCl3): d 25.6
(CH3), 26.9 (CH3), 33.3 (CH2CH2O), 65.1 (CH2CH2O), 69.4
(CH2OC(CH3)2), 73.0 (CH2CHCH2), 108.9 (C(CH3)2), 114.6, 130.0,
7. Experimental
7.1. General
NMR spectra were recorded on a Varian Gemini 2000 spectrom-
eter at 300 MHz for 1H, 75 MHz for 13C, and 121.5 MHz for 31P with

9944
A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
131.9, 163.8 (aryl), 190.7 (CHO). HRMS (ESI) m/z Calcd for
1H, CHHOH), 4.06 (m, 1H, CHHOH), 4.41 (m, 2H, CH2O), 4.73,
5.16 (2 br s, 2H, 2ꢁ OH), 7.23 (d, 1H, J = 7.8 Hz, aryl), 7.61–
7.78 (m, 7H, aryl), 8.09 (d, 1H, J = 8.1 Hz, aryl), 8.36 (d, 1H,
J = 7.8 Hz, aryl), 8.61 (m, 1H, aryl), 8.88 (m, 2H, aryl), 9.24 (d,
1H, J = 8.1 Hz, aryl), 13.49 (br s, 1H, NH). 13C NMR (DMSO-d6):
C14H18O4Na+ (MNa+) 273.1097. Found: 273.1101.
7.1.3. (S)-4-(2-(2,2-Dimethyl-1,3-dioxolan-4-yl)ethoxy)-1-
naphthaldehyde (3b)
Yield 4.8 g (92%) as an oil; Rf 0.31 (50% petroleum ether/diethyl
ether). 1H NMR (CDCl3): d 1.39 (s, 3H, CH3), 1.44 (s, 3H, CH3), 2.23
(m, 2H, CH2CH2O), 3.74 (dd, 1H, J = 7.2, 8.1 Hz, CHH), 4.21 (m,
1H,CH), 4.39 (m, 3H, CH2CH2O, CHH), 6.93 (d, 1H, J = 8.1 Hz, aryl),
7.57–7.60 (m, 1H, aryl), 7.68–7.71 (m, 1H, aryl), 7.90 (d, 1H,
J = 8.1 Hz, aryl), 8.31 (d, 1H, J = 9.0 Hz, aryl), 9.31(d, 1H, J = 9.0 Hz,
aryl), 10.20 (s, 1H, CHO). 13C NMR (CDCl3): d 25.7 (CH3), 27.0
(CH3), 33.4 (CH2CH2O), 65.5 (CH2CH2O), 69.5 (CH2OC(CH3)2), 73.2
(CH2CHCH2), 103.6 (aryl), 109.1 (C(CH3)2), 122.2, 124.9, 125.0,
125.4, 126.7, 129.5, 131.9, 139.6, 159.9 (aryl), 192.2 (CHO). HRMS
(ESI) m/z Calcd for C18H20O4Na+ (MNa+) 323.1254. Found:
323.1264.
d 33.1 (CH2CH2O), 65.2 (CH2CH2O), 66.1 (CHCH2OH), 68.2
(CHCH2OH), 104.6, 120.0, 121.9, 122.0–131.7 (aryl), 149.6
+
(C@N, aryl), 155.3 (aryl). HRMS (ESI) m/z Calcd for C29H25N2O3
(MH+) 449.1860. Found: 449.1864.
7.1.8. General procedure for preparation of 7 by DMT-
protection
(S)-4-(4-(1H-Phenanthro[9,10-d]imidazol-2-yl)phenoxy)butane-
1,2-diol (6a, 1.0 g, 2.5 mmol) or (S)-4-(4-(1H-phenanthro[9,10-
d]imidazol-2-yl)naphalen-1-yloxy)butane-1,2-diol (6b, 0.50 g, 1.11
mmol) was dissolved in anhydrous pyridine (20 ml). 4,40-Dime-
thoxytrityl chloride (1.2 equiv) was added under a nitrogen atmo-
sphere, and the reaction mixture was stirred at room temperature
for 24 h. The reaction was quenched by addition of MeOH (2 ml) fol-
lowed by addition of EtOAc (75 ml), and extracted with saturated
aqueous NaHCO3 (2ꢁ 20 ml). The H2O phase was extracted with
EtOAc (2ꢁ 10 ml), and the combined organic phases were dried
(Na2SO4), 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 silica gel column chromatography
[NEt3 (0.5%, v/v)/EtOAc (40–50%)/cyclohexane] to afford the DMT-
protected diols 7a,b.
7.1.4. General procedure for preparation of the phenanthroimi-
dazol compounds 6
Phenanthrene-9,10-dione (1 equiv) and ammonium acetate
(16.5 equiv) were dissolved in hot glacial acetic acid (10 ml). While
the mixture was stirred, a solution of (S)-4-(2-(2,2-dimethyl-1,3-
dioxolan-4-yl)ethoxy)benzaldehyde (3a, 2.0 g, 8.0 mmol) or (S)-4-
(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)-1-naphthaldehyde (3b,
1.0 g, 3.3 mmol) in 10 ml of glacial acetic acid was added dropwise.
The mixture was heated at 90 °C for 3 h and was then poured in to
water (200 ml). The mixture was neutralized with aqueous ammo-
nia to pH 7 and then cooled to room temperature. The precipitate
was filtered off and washed with large portions of H2O. The residue
was purified by silica gel column chromatography [MeOH/CH2Cl2
(1:1, v/v)] afforded 5 and 6a. Compound 6b was obtained directly
from the precipitate without using chromatography. Recrystalliza-
tion from toluene and one drop of NEt3.
7.1.9. (S)-4-(4-(1H-Phenanthro[9,10-d]imidazol-2-yl)phenoxy)-
1-(bis(4-methoxyphenyl) (phenyl)methoxy)butan-2-ol (7a)
Yield 1.4 g (79%) as a foam; Rf 0.43. 1H NMR (CDCl3): d 1.85 (m,
2H, CH2CH2O), 3.18 (m, 2H, CH2ODMT), 3.72 (s, 6H, 2ꢁ OCH3), 3.89
(m, 2H, CH2CH2O), 4.04 (m, 1H, CHOH), 6.66 (d, 2H, J = 8.4 Hz, aryl),
6.77 (d, 4H, J = 8.7 Hz, DMT), 7.17–7.30 (m, 9H, aryl), 7.40 (d, 2H,
J = 7.2 Hz, aryl), 7.55 (m, 4H, aryl), 7.88 (d, 2H, J = 8.4 Hz, aryl),
8.44 (br s, 1H, NH), 8.69 (m, 2H, aryl). 13C NMR (CDCl3): d 33.0
(CH2CH2O), 55.2 (2ꢁ OCH3), 64.7 (CH2CH2O), 67.4 (CHOH), 68.4
(CH2ODMT), 86.2 (OCPh3), 113.1, 114.7, 122.7–130.0, 135.9,
144.8, 149.6, 158.5, 159.7 (aryl). HRMS (ESI) m/z Calcd for
7.1.5. (S)-2-(4-(2-(2,2-Dimethyl-1,3-dioxolan-4-yl)ethoxy)phen-
yl)-1H-phenanthro[9,10-d] imidazole (5)
Yield 0.30 g (8.5%) as solids; Rf 0.55 (50% MeOH/CH2Cl2); mp
196–198 °C. 1H NMR (CDCl3): d 1.35 (s, 3H, CH3), 1.41 (s, 3H,
CH3), 1.91 (m, 2H, CH2CH2O), 3.55 (m, 1H, CHH), 3.82 (m, 2H,
CHH, CH2CHHO), 4.05 (m, 1H, CH2CHHO), 4.18 (m, 1H, CH), 6.64
(d, 2H, J = 8.7 Hz, aryl), 7.54 (m, 4H, aryl), 7.89 (d, 2H, J = 8.7 Hz,
aryl), 8.43 (br s, 2H, aryl), 8.67 (m, 2H, aryl). 13C NMR (CDCl3): d
25.7 (CH3), 26.9 (CH3), 33.3 (CH2CH2O), 64.5 (CH2CH2O), 69.5
(CH2OC(CH3)2), 73.3 (CH2CHCH2), 108.8 (C(CH3)2), 114.5, 121.7,
122.7–128.2 (aryl), 149.35 (C@N, aryl), 159.7 (aryl). HRMS (MALDI)
+
C46H41N2O5 (MH+) 701.3010. Found: 701.3044.
7.1.10. (S)-4-(4-(1H-Phenanthro[9,10-d]imidazol-2-yl)naphtha-
len-1-yloxy)-1-(bis(4-methoxy phenyl)(phenyl)methoxy)butan-
2-ol (7b)
Yield 0.47 g (56%) as a foam; Rf 0.34. 1H NMR (CDCl3): d 1.90 (m,
2H, CH2CH2O), 3.02 (br s, 1H, OH), 3.18 (m, 2H, CH2ODMT), 3.75 (s,
6H, 2ꢁ OCH3), 3.93 (m, 2H, CH2 CH2O), 4.07 (m, 1H, CHOH), 6.33
(m, 1H, aryl), 7.76 (d, 4H, J = 8.4 Hz, DMT), 7.18ꢂ7.55 (m, 18H,
aryl), 8.04 (d, 1H, J = 7.5 Hz, aryl), 8.55 (d, 1H, J = 7.5 Hz, aryl),
8.69 (m, 2H, aryl), 11.31 (br s, 1H, NH). 13C NMR (CDCl3): d 33.1
(CH2CH2O), 55.2, 55.2 (2ꢁ OCH3), 64.8 (CH2 CH2O), 67.5 (CHOH),
68.5 (CH2ODMT), 86.2 (OCPh3), 103.7, 113.1, 120.2, 122.0, 125.1–
130.0, 132.1, 135.9, 144.8 (aryl), 149.5 (C@N, aryl), 155.5, 158.4
(aryl). HRMS (ESI) m/z Calcd for C50H42N2O5Na+ (MNa+) 773.2987.
Found: 773.3003.
+
m/z Calcd for C28H27N2O3 (MH+) 439.2016. Found: 439.2002.
7.1.6. (S)-4-(4-(1H-Phenanthro[9,10-d]imidazol-2-yl)phenoxy)-
butane-1,2-diol (6a)
Yield 2.3 g (72%) as solids; Rf 0.10 (50% MeOH/CH2Cl2); mp 263–
265 °C. 1H NMR (DMSO-d6): d 1.77 (m, 1H, CHHCH2O), 2.04 (m, 1H,
CHHCH2O), 3.42 (m, 2H, CHHOH and CHOH), 3.76 (m, 1H, CHHOH),
4.23 (m, 2H,CH2CH2O), 4.69, 4.76 (2s, 2H, 2ꢁ OH), 7.20 (d, 2H,
J = 8.7 Hz, aryl), 7.63 (m, 2H, aryl), 7.75 (m, 2H, aryl), 8.30 (d, 2H,
J = 8.7 Hz, aryl), 8.61 (d, 2H, J = 8.1 Hz, aryl), 8.83 (d, 2H,
J = 8.1 Hz, aryl), 13.32 (br s, 1H, NH). 13C NMR (DMSO-d6): d 33.1
(CH2CH2O), 64.8 (CH2CH2O), 66.0 (CHCH2OH), 68.1 (CHCH2OH),
114.8, 121.9, 122.8–127.7 (aryl), 149.4 (C@N, aryl), 159.7 (aryl).
HRMS (MALDI) m/z Calcd for C25H23N2O3+ (MH+) 399.1703. Found:
399.1689.
7.1.11. General procedure for preparation of phosphoramidite
(8)
DMT-protected compound 7a (0.4 g, 0.57 mmol) or 7b (0.1 g,
0.17 mmol) was dissolved under an argon atmosphere in anhy-
drous CH2Cl2 (10–15 ml). N,N0-Diisopropylammonium tetrazolide
(1.5 equiv) was added, followed by dropwise addition of 2-cyano-
ethyl N,N,N0,N0-tetraisopropylphosphordiamidite (3 equiv) under
external cooling with an ice-water bath. The reaction mixture
was stirred at room temperature overnight. After 24 h, analytical
TLC showed no more starting material and the reaction was
7.1.7. (S)-4-(4-(1H-Phenanthro[9,10-d]imidazol-2-yl)naphalen-
1-yloxy)butane-1,2-diol (6b)
Yield 1.2 g (80%) as solids; mp 165–167 °C. 1H NMR (DMSO-
d6): d 2.05 (m, 2H, CH2CH2O), 3.61 (m, 1H, CHOH), 3.85 (m,

A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
9945
quenched 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 washed with CH2Cl2 (25 ml), the organic phase
was dried (Na2SO4) and filtered, and the solvents were evaporated
in vacuo. The residue was purified by silica gel column chromatog-
raphy [NEt3 (0.5%, v/v)/EtOAc (40–50%)/cyclohexane] to afford the
final products 8a,b as a foam, which were used in DNA synthesis
after drying under diminished pressure.
(CH2OH), 68.1 (CHOH), 104.2, 120.4, 121.5, 125.4, 126.8, 128.1,
129.8, 131.2, 134.8, 145.4, 154.3 (aryl). HRMS (MALDI) m/z Calcd
for C17H18N2O3Na+ (MNa+) 321.1210. Found: 321.1217.
7.1.16. (S)-4-(4-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-
hydroxybutoxy)-1-naphthaldehyde (11)
Compound 9 (0.50 g, 1.92 mmol) was dissolved in dry pyridine
(20 ml) and 4,40-dimethoxytrityl chloride (DMT-Cl) (0.78 g,
2.30 mmol) was added under a nitrogen atmosphere. The reaction
mixture was stirred for 24 h at room temperature. The solvent was
evaporated off under reduced pressure, and the residue was puri-
fied by silica gel column chromatography [NEt3 (0.5%, v/v)/EtOAc
(30–50%)/cyclohexane] affording compound 11. Yield 0.65 g
(60%) as a foam; Rf 0.21. 1H NMR (CDCl3): d 2.08 (m, 2H, CH2CH2O),
2.49 (s, 1H, OH), 3.21, 3.32 (2ꢁ m, 2H, CH2ODMT), 3.76 (s, 6H, 2ꢁ
OCH3), 4.13 (m, 1H, CHOH), 4.34 (m, 2H, CH2CH2O), 6.80 (d, 4H,
J = 9.0 Hz, DMT), 6.86 (d, 1H, J = 8.1 Hz, aryl), 7.29 (m, 8H, aryl),
7.43 (d, 1H, J = 6.9 Hz, aryl), 7.56 (m, 1H, aryl), 7.72 (m, 1H, aryl),
7.89 (d, 1H, J = 8.1 Hz, aryl), 8.22 (d, 1H, J = 8.4 Hz, aryl), 9.30 (d,
1H, J = 8.4 Hz, aryl), 10.19 (s, 1H, CHO). 13C NMR (CDCl3): d 32.9
(CH2CH2O), 55.2 (2ꢁ OCH3), 65.3 (CH2CH2O), 67.4 (CHOH), 68.2
(CH2ODMT), 86.3 (OCPh3), 103.7, 113.2, 122.3–130.0, 131.9,
135.8, 139.7, 144.7, 158.5, 160.0 (aryl), 192.3 (CHO). HRMS (ESI)
m/z Calcd for C36H34O6Na+ (MNa+) 585.2248. Found: 585.2253.
7.1.12. (S)-4-(4-(1H-Phenanthro[9,10-d]imidazol-2-yl)pheno-
xy)-1-(bis(4-methoxyphenyl)(phenyl)-methoxy)butan-2-yl 2-
cyanoethyl diisopropylphosphoramidite (8a)
Yield 0.44 g (86%) as a foam; Rf 0.68. 13C NMR (CDCl3): d 20.1
(CH2CN), 24.4, 24.5, 24.6, 24.7 (2ꢁ CH(CH3)2), 33.0 (CH2CH2O),
43.1, 43.2 (2ꢁ C(CH3)2), 55.2 (2ꢁ OCH3), 57.8 (OCH2CH2CN), 64.1
(CH2CH2O), 66.4 (CHOP [NPr2]2), 69.4 (CH2ODMT), 86.0 (OCPh3),
113.0, 114.9, 122.5–130.1, 136.1, 136.2, 144.9, 149.8, 158.4,
158.4, 160.0 (aryl). 31P NMR (CDCl3): d 149.98, 150.05 in a 5:4 ratio.
HRMS (ESI) m/z Calcd for C55H57N4O6PNa+ (MNa+) 923.3909.
Found: 923.3913.
7.1.13. (S)-4-(4-(1H-Phenanthro[9,10-d]imidazol-2-yl)naphtha-
len-1-yloxy)-1-(bis(4-methoxy phenyl)(phenyl)methoxy)butan-
2-yl 2-cyanoethyl diisopropylphosphoramidite (8b)
Yield 0.11 g (81%) as a foam; Rf 0.64. 13C NMR (CDCl3): d 20.08
(CH2CN), 24.4, 24.5, 24.6, 24.7 (2ꢁ CH(CH3)2), 33.0 (CH2CH2O),
43.1, 43.3 (2ꢁ CH(CH3)2), 55.2 (2ꢁ OCH3), 57.9 (OCH2CH2CN),
64.2 (CH2CH2O), 66.4 (CHOP[NPr2]2), 70.8 (CH2ODMT), 86.1
(OCPh3), 104.0, 113.1, 117.7, 120.6–132.5, 136.1, 136.2, 145.0,
149.5, 155.8, 158.4 (aryl). 31P NMR (CDCl3): d 149.98, 150.48 in a
2:1 ratio. HRMS (ESI) m/z Calcd for C59H59N4O6PNa+ (MNa+)
973.4065. Found: 973.4021.
7.1.17. (S)-4-(4-(1H-Imidazol-2-yl)naphthalen-1-yloxy)-1-
(bis(4-methoxyphenyl)(phenyl)-methoxy)butan-2-ol (12)
To a solution of (S)-4-(4-(bis(4-methoxyphenyl)(phenyl)metho-
xy)-3-hydroxybutoxy)-1-naphthaldehyde (11) (0.44 g, 0.79 mmol)
in EtOH (1.1 ml) was added dry MeCN (5 ml) to give a clear solu-
tion. Forty percent of glyoxal in H2O (0.18 ml, 4.0 mmol) and
20 M ammonium hydroxide (0.27 ml) was added at 0 °C. The mix-
ture was stirred for 30 min at 0 °C and then at room temperature
under a nitrogen atmosphere overnight. The reaction mixture
was concentrated in vacuo and the residue was purified by silica
gel column chromatography [EtOAc/cyclohexane/NEt3 (90:8:2, v/
v/v)] affording compound 12. Yield 0.15 g (32%) as a foam; Rf
0.50. 1H NMR (CDCl3): d 1.94 (m, 2H, CH2CH2O), 3.19, 3.29 (2ꢁ
m, 2H, CH2ODMT), 3.74 (s, 6H, 2ꢁ OCH3), 4.11 (m, 4H, CH2CH2O
and CHOH), 6.55 (d, 1H, J = 8.1 Hz, aryl), 6.78 (d, 4H, J = 8.7 Hz,
DMT), 7.08 (s, 2H, imidazole), 7.19–7.31 (m, 7H, aryl), 7.41 (m,
5H, aryl), 8.16 (d, 1H, J = 9.0 Hz, aryl), 8.44 (d, 1H, J = 8.1 Hz, aryl).
7.1.14. (S)-4-(3,4-Dihydroxybutoxy)-1-naphthaldehyde (9)
Compound 3b (0.85 g, 2.83 mmol) was stirred in 80% acetic acid
(25 ml) for 24 h at room temperature. The solvent was removed in
vacuo, and the residue was coevaporated twice with toluene/EtOH
(30 ml, 5:1, v/v). The residue was dried in vacuo to afford 4-(3,4-
dihydroxybutoxy)-1-naphthaldehyde 9. Yield 0.74 g (100%) as an
oil which was used in the next step without further purification.
1H NMR (DMSO-d6): d 1.83 (m, 1H, CHHCH2O), 2.30 (m, 1H,
CHHCH2O), 3.42 (m, 2H, CH2CHOH , CHHOH), 3.80 (m, 1H, CHHOH),
4.42 (m, 2H, CH2CH2O), 4.63, 4.73 (s, 2H, 2ꢁ OH), 7.22 (m, 1H, aryl),
7.64 (m, 1H, aryl), 7.75 (m, 1H, aryl), 8.14 (d, 1H, J = 8.1 Hz, aryl),
8.31 (d, 1H, J = 7.8 Hz, aryl), 9.23 (d, 1H, J = 8.4 Hz, aryl), 10.18 (s,
1H, CHO). 13C NMR (DMSO-d6): d 32.8 (CH2CH2O), 65.7 (CH2CH2O),
65.9 (CH2OH), 68.0 (CHOH), 104.6, 122.1–131.1, 140.4, 159.6 (aryl),
192.7 (CHO). HRMS (ESI) m/z Calcd for C15H16O4Na+ (MNa+)
283.0941. Found: 283.0948.
13C NMR (CDCl3):
d
33.1 (CH2CH2O), 55.2 (2ꢁ OCH3), 64.8
(CH2CH2O), 67.5 (CHOH), 68.4 (CH2ODMT), 86.2 (OCPh3), 103.8,
113.1, 120.7, 122.1, 125.4–130.0, 132.0, 135.9, 136.0, 144.8,
146.4, 155.2, 158.4 (aryl). HRMS (ESI) m/z Calcd for C38H36N2O5Na+
(MNa+) 623.2517. Found: 623.2494.
7.1.18. (S)-4-(4-(1H-Imidazol-2-yl)naphthalen-1-yloxy)-1-
(bis(4-methoxyphenyl)(phenyl)-methoxy)butan-2-yl 2-
7.1.15. (S)-4-(4-(1H-Imidazol-2-yl)naphthalen-1-yloxy)butan-
1,2-diol (10)
cyanoethyl diisopropylphosphoramidite (13)
Compound 12 (0.10 g, 0.17 mmol) was dissolved under an ar-
gon atmosphere in anhydrous CH2Cl2 (10 ml). N,N0-Diisopropyl
ammonium tetrazolide (0.04 g, 0.25 mmol) was added, followed
by dropwise addition of 2-cyanoethyl tetraisopropylphosphordi-
amidite (0.15 g, 0.45 mmol) under external cooling with an ice-
water bath. The reaction mixture was stirred at room temperature
under an argon atmosphere overnight. After 24 h, analytical TLC
showed no more starting material. The solvent was evaporated un-
der reduced pressure and the residue was purified by silica gel col-
umn chromatography [EtOAc/cyclohexane/NEt3 (90:8:2, v/v/v)]
affording compound 13. Yield: 0.11 g (81%) as a foam; Rf 0.70.
13C NMR (CDCl3): d 20.2 (CH2CN), 24.4, 24.5, 24.6, 24.7 [2ꢁ
CH(CH3)2], 33.2 (CH2CH2O), 43.0, 43.2 [2ꢁ CH(CH3)2], 55.2 (2ꢁ
OCH3), 57.7 (OCH2CH2CN), 64.3 (CH2CH2O), 66.5 (CHOP[NPr2]2),
71.0 (CH2ODMT), 86.0 (OCPh3), 104.0, 113.0, 121.0, 122.2, 125.4–
To a solution of (S)-4-(3,4-dihydroxybutoxy)-1-naphthaldehyde
(9, 0.10 g, 0.38 mmol) in EtOH (0.54 ml) was added about dry
MeCN (3 ml) to give a clear solution. Forty percent of glyoxal in
H2O (0.10 ml, 1.93 mmol) and 20 M ammonium hydroxide
(0.13 ml) was added at 0 °C. The mixture was stirred for 30 min
at 0 °C and then at room temperature overnight. The mixture
was concentrated in vacuo and the residue was purified by silica
gel column chromatography [EtOAc/cyclohexane/NEt3 (90:8:2, v/
v/v)] to give compound 10. Yield 0.05 g (44%) as an oil; Rf 0.11.
1H NMR (DMSO-d6): d 2.04 (m, 2H, CH2CH2O), 3.42 (m, 2H, CHOH
and CHHOH), 3.80 (m, 1H, CHHOH), 4.36 (m, 2H, CH2CH2O), 4.69,
4.71 (2s, 2H, 2ꢁ OH), 6.70–8.01 (m, 6H, aryl), 8.27 (d, 1H,
J = 8.7 Hz, aryl), 9.01 (d, 1H, J = 8.7 Hz, aryl), 12.38 (br s, 1H, NH).
13C NMR (DMSO-d6): d 33.1 (CH2CH2O), 65.0 (CH2CH2O), 66.0

9946
A. M. A. Osman et al. / Bioorg. Med. Chem. 16 (2008) 9937–9947
130.1, 132.2, 136.1, 136.2, 144.9, 146.5, 155.3, 158.4 (aryl). 31P
NMR (CDCl3): d 149.99, 150.09 in a 4:3 ratio. HRMS (ESI) m/z Calcd
for C47H53N4O6PNa+ (MNa+) 823.3585. Found: 823.3581.
0.0 nm slit is not completely closed and allowed sufficient light
to pass for the measurement.
7.4. Molecular modeling
7.2. Oligonucleotide synthesis, purification, and melting
temperature determination
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
v97.2 from MSI, followed by incorporation of the modified
nucleotide.
DMT-on oligodeoxynucleotides were carried out at 0.2 lmol
scales on 500 Å CPG supports with an ExpediteTM Nucleic Acid Syn-
thesis System Model 8909 from Applied Biosystems with 1H-tetra-
zole as an activator for coupling reaction. The appropriate amidite
(8a,b and 13) was dissolved in dry CH2Cl2 and inserted into the
growing oligonucleotides chain using an extended coupling time
(10 min). DMT-on oligonucleotides bound to CPG supports were
treated with aqueous ammonia (32%, 1 ml) at room temperature
and then at 55 °C overnight. Purification of 50-O-DMT-on ONs
was accomplished by reversed-phase semipreparative HPLC on a
Waters Xterra MS C18 column with a Waters Delta Prep 4000 Pre-
parative Chromatography System (Buffer A [0.05M triethylammo-
nium acetate in H2O (pH 7.4)] and Buffer B (75% MeCN in H2O)).
Flow 2.5 ml minꢂ1. Gradients: 2 min 100% A, linear gradient to
70% B in 38 min, linear gradient to 100% B in 3 min and then
Acknowledgments
This work supported by the Sixth Framework Program Marie
Curie Host Fellowships for Early Stage Research Training under
contract number MEST-CT-2004-504018 and by the Nucleic Acid
Center, which is funded by The Danish National Research Founda-
tion for studies on nucleic acid chemical biology.
100% A in 10 min). ODNs were DMT deprotected in 100
lL 80% ace-
tic acid over 20 min. Afterwards, aqueous AcONa (1 M, 50
l
L) was
added and the ONs were precipitated from EtOH (96%). All modi-
fied ODNs were confirmed by MALDI-TOF analysis on a Voyager
Elite Bio spectroscopy Research Station from PerSeptive Biosys-
tems. ODN Found m/z (Calculated m/z): ON2 4589.3 (4589.2),
ON3 4580.1 (4581.3), ON4 4627.3 (4631.3), ON5 4476.5 (4481.1),
ON7 4579.1 (4581.3), ON8 4629.2 (4631.3), ON9 4479.5 (4481.1),
ON10 4591.7 (4581.3), ON11 4627.6 (4631.3), ON13 5042.7
(5040.7), ON14 5138.2 (5140.8), ON16 4578.9 (4581.3), and
ON17 4576.8 (4581.3). The purity of the final TFOs was found to
be over 90%, checked by ion-exchange chromatography using La-
Chrom system from Merck Hitachi on Genpak-Fax column
(Waters). Melting temperature measurments were performed on
a Perkin-Elmer UV/vis spectrometer Lambda 35 fitted with a PTP-
6 temperature programmer. The triplexes were formed by first
mixing the two strands of the Watson–Crick duplex, each at a con-
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