Synthesis of a new intercalating nucleic acid 6H-indolo[2,3-b] quinoxaline oligonucleotides to improve thermal stability of Hoogsteen-type triplexes

Article abstract of DOI:10.1080/15257770.2013.765013

A new intercalating nucleic acid monomer X was obtained in high yield starting from alkylation of 4-iodophenol with (S)-(+)-2-(2,2-dimethyl-1,3- dioxolan-4-yl)ethanol under Mitsunobu conditions followed by hydrolysis with 80% aqueous acetic acid to give a diol which was coupled under Sonogashira conditions with trimethylsilylacetylene (TMSA) to achieve the TMS protected (S)-4-(4-((trimethylsilyl)ethynyl)phenoxy)butane-1,2-diol. Tetrabutylammonium flouride was used to remove the silyl protecting group to obtain (S)-4-(4-ethynylphenoxy)butane-1,2-diol which was coupled under Sonogashira conditions with 2-(9-bromo-6H-indolo[2,3-b]quinoxalin-6-yl)-N,N- dimethylethanamine to achieve (S)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[2, 3-b]quinoxalin-9-yl)ethynyl)phenoxy)butane-1,2-diol. This compound was tritylated with 4,4′-dimethoxytrityl chloride followed by treatment with 2-cyanoethyltetraisopropylphosphordiamidite in the presence of N,N′-diisopropyl ammonium tetrazolide to afford the corresponding phosphoramidite. This phosphoramidite was used to insert the monomer X into an oligonucleotide which was used for thermal denaturation studies of a corresponding parallel triplex.

Full text of DOI:10.1080/15257770.2013.765013

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Synthesis of A New Intercalating Nucleic
Acid 6H-INDOLO[2,3-b] Quinoxaline
Oligonucleotides to Improve Thermal
Stability Of Hoogsteen-Type Triplexes
Amany M. A. Osman a b , Erik B. Pedersen a & Jan Bergman c
a Department of Physics, Chemistry and Pharmacy, Nucleic Acid
Center, University of Southern Denmark, Odense, Denmark
b Department of Chemistry, Faculty of Science, Menoufia University,
Shebin El-Koam, Egypt
c Department of Biosciences and Nutrition, Unit for Organic
Chemistry, Karolinska Institute, Huddinge, Sweden
Published online: 28 Feb 2013.
To cite this article: Amany M. A. Osman , Erik B. Pedersen & Jan Bergman (2013): Synthesis of A
New Intercalating Nucleic Acid 6H-INDOLO[2,3-b] Quinoxaline Oligonucleotides to Improve Thermal
Stability Of Hoogsteen-Type Triplexes, Nucleosides, Nucleotides and Nucleic Acids, 32:2, 98-108
To link to this article: http://dx.doi.org/10.1080/15257770.2013.765013
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Nucleosides, Nucleotides and Nucleic Acids, 32:98–108, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.765013
SYNTHESIS OF A NEW INTERCALATING NUCLEIC ACID
6H-INDOLO[2,3-b] QUINOXALINE OLIGONUCLEOTIDES TO IMPROVE
THERMAL STABILITY OF HOOGSTEEN-TYPE TRIPLEXES
Amany M. A. Osman,1,2 Erik B. Pedersen,1 and Jan Bergman3
1Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, University of
Southern Denmark, Odense, Denmark
2Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koam, Egypt
3Department of Biosciences and Nutrition, Unit for Organic Chemistry, Karolinska Institute,
Huddinge, Sweden
A new intercalating nucleic acid monomer X was obtained in high yield starting from alky-
2
lation of 4-iodophenol with (S)-(+)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol under Mitsunobu
conditions followed by hydrolysis with 80% aqueous acetic acid to give a diol which was cou-
pled under Sonogashira conditions with trimethylsilylacetylene (TMSA) to achieve the TMS pro-
tected (S)-4-(4-((trimethylsilyl)ethynyl)phenoxy)butane-1,2-diol. Tetrabutylammonium flouride was
used to remove the silyl protecting group to obtain (S)-4-(4-ethynylphenoxy)butane-1,2-diol which
was coupled under Sonogashira conditions with 2-(9-bromo-6H-indolo[2,3-b]quinoxalin-6-yl)-N,N-
dimethylethanamine to achieve (S)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[2,3-b]quinoxalin-
9-yl)ethynyl)phenoxy)butane-1,2-diol. This compound was tritylated with 4,4ꢁ-dimethoxytrityl chlo-
ride followed by treatment with 2-cyanoethyltetraisopropylphosphordiamidite in the presence of N,Nꢁ-
diisopropyl ammonium tetrazolide to afford the corresponding phosphoramidite. This phospho-
ramidite was used to insert the monomer X into an oligonucleotide which was used for thermal
denaturation studies of a corresponding parallel triplex.
Keywords Intercalating nucleic acid; 6H-indolo[2,3-b]quinoxaline; hoogsteen base
pairing; thermal stability; triplex forming oligonucleotides
INTRODUCTION
There is an interest for high-affinity and sequence specific recognition
of double stranded DNA for diagnostic and therapeutic purposes.[1,2] There-
fore, there is an interest in the design and synthesis of new triplex forming
Received 21 November 2012; accepted 7 January 2013.
This work supported by Partnership and Ownership Initiative (ParOwn Fellowship 1210) launched
by Egyptian Ministry of Higher Education and State for Scientific Research.
Address correspondence to Erik B. Pedersen, Department of Physics, Chemistry and Pharmacy,
University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark. E-mail: erik@sdu.dk
98

Nucleic Acid 6H-Indolo[2,3-b] Quinoxaline Oligonucleotides
99
oligonucleotides (TFOs) that can hybridize efficiently and selectively to dou-
ble stranded DNA at neutral pH. The so formed DNA triple helix consists
of a third strand of nucleic acid of appropriate sequence bound in the
major groove of an intact duplex.[3] These structures form most readily in
polypurine.polypyrimidine tracts and they are stabilized by hydrogen bonds
between the third strand bases and the duplex purines.[4] Depending on
the target sequence the TFO might consists of purines or pyrimidines. In
the pyrimidine motif, a (C,T) containing oligonucleotides binds parallel
to the target oligopurine strand through Hoogsteen hydrogen bonds, and
forms canonical C.GxC+ and T.AxT base triplets. Due to the requirement
for cytosine protonation, the stability of a C.GxC+ motif triple helix is pH
dependent. In the purine motif, the (G,A) containing oligonucleotide binds
antiparallel to the oligopurine strand of the duplex by forming C.GxG and
T.AxA base triplets through reverse Hoogsteen hydrogen bonds.[5–10] The
insertions of conjugated intercalators in the TFO have been shown to be
an effective way to stabilize the triplexes. The twisted intercalating nucleic
acids (TINA) inserted as a bulge in a triple-helix forming oligonucleotidde
(TFO), stabilize parallel Hoogsteen triplexes (Figure 1).[11] In addition,
Paramavisam et al.[12] have recently shown that incorporation of TINA
monomers in G-rich oligonucleotides interferes with quadruplex formation
at physiological potassium concentrations releasing the oligonucleotide for
antiparallel triplex formation.
Heterocyclic rings with nitrogen atoms constitute the core structures
of molecules having various biological activities. The chemistry of indolo-
quinoxalines is of considerable interest possessing a broad spectrum of
biological activities. Derivatives of indolo[2,3-b]quinoxaline possess diverse
FIGURE 1 Intercalating nucleic acids TINA and 5ꢁ-DNA-X-DNA and the intercalators ellipticine and
B-220.

100
A. M. A. Osman et al.
biological activities such as anticancer, anticonvulsant and antibacterial.
However, several condensed ring systems containing indole have antiviral ac-
tivity.[13] Such compounds are distantly related to ellipticine and quinacrines,
which are known to intercalate in DNA.[14] Intercalation of planar aro-
matic molecules was discovered in the early 1960s, when it was observed
that acridine derivatives have the capacity to insert between two consecu-
tive DNA base pairs.[15–17] Rapidly, this mode of binding to nucleic acids
was found to occur with many compounds, in particular dyes like ethid-
ium bromide and methylene blue. Later, it was realized that drugs could
also exert a therapeutic effect via intercalation into DNA. Nowadays, numer-
ous intercalating agents are being used as therapeutics for the treatment
of cancer as well as for parasitic and viral diseases.[18–20] For example, the
antitumor drugs Daunomycin, mitoxantrone and amsacrine contain a pla-
nar chromophore that intercalates into DNA. 6H-Indolo[2,3-b]quinoxaline
compounds represent an important series of DNA intercalating agents en-
dowed with antiviral and cytotoxic activities.[21] Their effect on herpes viruses
was assayed in different cells. For example, compound B-220 (2,3-dimethyl-
6-(2-dimethylaminoethyl)-6H-indolo[2,3-b]quinoxaline) has shown remark-
able activity against herpes virus.[22, 23] Both the dimethylaminoethyl side
chain and the tetracyclic chromophore contribute to the interaction with
DNA.[24–26] Compound B-220 has a structural analogy with the antitumor
alkaloid ellipticine which is a well-known DNA intercalator.[27] In our ongo-
ing research, we are looking for new analogues of intercalating nucleic acids
and we considered it interesting to replace the pyrene moiety in TINA with a
moiety identical to the compound B-220 because this moiety is most likely a
good intercalator and also because compound B-220 itself shows low in vivo
toxicity when tested in rats.[22]
RESULTS AND DISCUSSION
Chemistry
The synthetic route for obtaining the desired acyclic amidite 11 is shown
in (Scheme 1). The synthesis of (S)-4-(4-iodophenoxy)butane-1,2-diol 4
was achieved under Mitsunobu conditions as previously described.[28] In
this way, (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol 1, diethyl azodicar-
boxylate (DEAD), triphenylphosphine, and 4-iodophenol 2 were reacted
in tetrahydrofuran (THF) to afford (S)-4-(2-(4-iodophenoxy)ethyl)-2,2-
dimethyl-1,3-dioxolane 3. Subsequent treatment with 80% aqueous acetic
acid gave the diol 4 in satisfactory yield which was used in the next
step without further purification. The subsequent Sonogashira coupling
with trimethylsilylacetylene (TMSA) was done with Pd(PPh3)2Cl2, CuI,

Nucleic Acid 6H-Indolo[2,3-b] Quinoxaline Oligonucleotides
101
I
O
O
I
I
O
O
HO
HO
DEAD/THF
Ph3P
80% CH3COOH/H2O
+
OH
O
O
OH
3
1
2
4
TMS
H
HO
HO
HO
HO
Bu4N+F-/THF/30min/0oC
NH4Cl/H2O
Si
Pd(PPh3)2Cl2/CuI
NEt3
4
+
C
O
O
CH
7
6
5
N
N
N
N
N
N
N
N
N
DMT-Cl
pyridine
Pd(PPh3)2Cl2/CuI
NEt3
7
+
N
N
N
Br
8
O
O
DMTO
OR
HO
OH
9
R = H
11 R = P(NiPr2)O(CH2)2CN
10
NC(CH2)2OP(NiPr2)2
CH2Cl2, r.t.
SCHEME 1
NEt3, under Ar affording the TMS protected (S)-4-(4-((trimethylsilyl)-
ethynyl)phenoxy)butane-1,2-diol 6 in 95% yield. The silyl protecting group
was removed by addition of tetrabutylammonium flouride (TBAF) in dry
THF to obtain (S)-4-(4-ethynylphenoxy)butane-1,2-diol 7 in 81% yield,
which was coupled under Sonogashira conditions with 2-(9-bromo-6H-
indolo[3,2-b]quinoxalin-6-yl)-N,N-dimethylethanamine
8
prepared by
method of Bergman and Akerfeldt [29], Pd(PPh3)2Cl2, CuI, and NEt3
under Ar in a micro wave cavity to achieve (S)-4-(4-((6-(2-(dimethylamino)
ethyl)-6H-indolo[3,2-b]quinoxalin-9-yl)ethynyl)phenoxy) butane-1,2-diol
9 in 89% yield. The primary hydroxyl group was protected with 4,4ꢁ-
dimethoxytrityl chloride in dry pyridine to give (S)-1-(bis(4-methoxyphenyl)
(phenyl)methoxy)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[3,2-b]qu-
inoxalin-9-yl)ethynyl)phenoxy)butan-2-ol 10 in 52% yield. Finally, the
corresponding phosphoramidite 11 was obtained in 87% yield after treat-
ment with 2-cyanoethyl N,N,Nꢁ,Nꢁ-tetraisopropylphosphordiamidite in the
presence of diisopropylammonium tetrazolide in dry CH2Cl2. The obtained
phosphoramidite 11 was incorporated into a 14-mer oligonucleotide by
a standard phosphoramidite protocol on an automated DNA synthesizer.
˚

102
A. M. A. Osman et al.
However, an extended coupling time (10 minutes) in the oligonucleotide
synthesis as was used for the amidite 11. The modified ODN was purified
by reversed-phase HPLC, and its mass was confirmed by MALDI-TOF-MS
analysis. The purity of the final sequence was determined by ion-exchange
HPLC (IE-HPLC) to be more than 95%.
Thermal Stability Studies
The thermal stability of parallel triplex containing the intercalating
monomer X was evaluated by thermal denaturation experiments. The melt-
ing temperatures 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 oligonu-
cleotides toward the duplex (D1).[30] was assessed both at pH 6.0 and pH
7.2, the ultimate goal being to find parallel Hoogsteen-type triplex at physi-
ological pH conditions. Stabilization of parallel Hoogsteen-type triplex was
found in ON2 and compared with the wild type ON1 at pH 6.0 and pH
7.2. For the incorporation of the intercalating monomer X in the 14-mer
oligonucleotide at pH 6.0, the triplex stability of ON2/D1 (Tm = 39.8◦C)
is increased compared to the unmodified triplex ON1/D1 (Tm = 26.5◦C)
(Table 1 and Figure 2). For the incorporation of the intercalating monomer
X at pH 7.2, the triplex stability of ON2/D1 (Tm = 26.9◦C) is increased
compared to the unmodified triplex ON1/D1 (Tm < 5.0◦C). In both
cases, the stabilization confirms that the intercalating properties of X con-
tribute to stabilize the triplexes. Although the stabilization is lower than
the one previously observed for TINA, we find it useful to add a new in-
tercalator to the library of intercalating nucleic acids for studies of their
bioactivity.[12]
TABLE 1 Tm (◦C) data for parallel triplex, evaluated from UV melting curves (λ = 260 nm)
3ꢁ-CTGCCCCTTTCTTTTTT-5ꢁ
5ꢁ-GACGGGGAAAGAAAAAA-3ꢁ (D1)
Entry
TFOa
Tm (◦C) pH 6.0
Tm (◦C) pH 7.2
ON1
ON2
ON3b
5ꢁ-CCCCTTTCTTTTTT-3ꢁ
5ꢁ-CCCCTTXTCTTTTTT-3ꢁ
5ꢁ-CCCCTTPTCTTTTTT-3ꢁ
26.5
39.8
45.5
<5.0
26.9
28.0
aC = 1.5 μM of ON1–ON2 and 1.0 μM of each strand of dsDNA (D1) in 20 mM sodium cacodylate,
100 mM NaCl, 10 mM MgCl2, pH 6.0, and 7.2. Duplex Tm = 56.7◦C (pH 6.0) and 56.2◦C (pH 7.2). Tm
values determined at 260 nm.
bP = TINA, data taken from;[31]
.

Nucleic Acid 6H-Indolo[2,3-b] Quinoxaline Oligonucleotides
103
FIGURE 2 First derivatives plots of triplex melting for ON1 wild-type triplex and ON2 incorporating
monomer X recorded at 260 nm versus increasing temperature (1◦C/minute) in 20 mM sodium cacody-
late, 100 mM NaCl, 10 mM MgCl2 at pH 6.0.
CONCLUSIONS
We have synthesized a new intercalating nucleic acid X and shown its
incorporation into 14-mer oligonucleotides using standard oligonucleotide
synthesis procedures. Thermal melting studies showed that the intercalator
X upon incorporation into a 14-mer oligonucleotide increase the thermal
stability of a Hoogsteen-type triplex.
EXPERIMENTAL SECTION
General Information
NMR spectra were recorded on a Varian Gemini 2000 spectrometer at
1
300 MHz for H, 75 MHz for 13C and 121.5 MHz for 31P with TMS as an
1
internal standard for H NMR, deuterated solvents CDCl3 (δ 77.00 ppm),
DMSO-d6 (δ 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 Transform Ion Cyclotron Resonance Mass Spec-
trometer (IonSpec, Irvine, CA). For accurate ion mass determinations, the
(MH+) or (MNa+) ion was peak matched using ions derived from the
2,5-dihydroxybenzoic acid matrix. Electrospray ionization mass spectra (ESI-
MS) were performed on a 4.7 T HiResESI Uitima (FT) mass spectrometer.
Both spectrometers are controlled by the OMEGA Data System. Melting
points were determined on a Bu¨chi melting point apparatus. Silica gel
(0.040–0.063 mm) used for column chromatography and analytical silica

104
A. M. A. Osman et al.
gel TLC plates 60 F254 were purchased from Merck. Solvents used for col-
umn chromatography were distilled prior to use, while reagents were used
as purchased.
Synthesis
(S)-4-(4-((trimethylsilyl)ethynyl)phenoxy)butane-1,2-diol (6)
To the solution of (S)-4-(4-Iodophenoxy)butane-1,2-diol 4 (0.5 g,
1.62 mmol), Pd(PPh3)2Cl2 (81 mg, 0.12 mmol), and CuI (36 mg, 0.19 mmol)
were dissolved in dry NEt3 (20 mL). The reaction mixture was flushed with
Ar for 10 minutes before TMSA 5 (0.32 g, 3.24 mmol) was added. The reac-
tion mixture was stirred under Ar atmosphere for 24 hours. The solvent was
removed under reduced pressure and the residue was dissolved in CH2Cl2
(50 mL), which was washed with 0.3 M aq. EDTA (2 × 50 mL). After back
extraction with CH2Cl2 (50 mL), the organic phase was dried (MgSO4), fil-
tered, and concentrated under reduced pressure. The residue was purified
by column chromatography (silica gel; CH2Cl2–MeOH 5%, v/v) to afford
compound 6 as a brown oil. Yield 0.53 g (95%). 1H NMR (300 MHz; CDCl3):
δ 0.19, 0.24, 0.26 (3 s, 9H) 1.90–1.96 (m, 2H), 2.82 (br.s, 2H), 3.50–3.56
(m, 1H), 3.69, 3.73 (dd, J = 3.3 Hz, 1H), 3.97–3.99 (m, 1H), 4.10–4.16 (m,
2H), 6.81 (d, J = 9.0 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H) ppm. 13C NMR
(75 MHz; CDCl3): δ 0.00, 0.03, 0.07, 32.5, 65.0, 66.6, 69.8, 85.9, 92.6, 114.3,
115.6, 133.6, 158.7 ppm. EI-MS: Calcd for C15H22O3Si [M]+ 278.42, found:
278.13.
(S)-4-(4-ethynylphenoxy)butane-1,2-diol (7)
Dissolve compound 6 (0.5 g, 1.8 mmol) in dry THF (15 ml) and added
1.2 eq. TBAF tetrabutylammoniumflouride (2.16 ml, 2.16 mmol) slowly to
this solution. Cooled at 0◦C. The resulting solution was stirred at this temper-
ature for 30 minutes. The reaction was quenched by addition of saturated
aq. NH4Cl and the mixture extracted with EtOAC (30 ml). After back ex-
traction with EtOAC (30 ml), the combined organic phase was washed with
water (2 × 50 ml) and brine (2 × 50 ml), dried (MgSO4), filtered and
concentrated under reduced pressure. The residue was purified by column
chromatography (silica gel; 5% MeOH in CH2Cl2, v/v) to afford compound
7 as brown oil. Yield 0.30 g (81%). 1H NMR (300 MHz; CDCl3): δ 1.87–1.93
(m, 2H), 3.01 (s, 1H), 3.53–3.55 (m, 1H), 3.65–3.72 (m, 3H), 4.09– 4.14
(m, 1H), 6.83 (d, J = 8.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H) ppm. 13C NMR
(75 MHz; CDCl3): δ 32.5, 65.0, 66.7, 69.4, 75.7, 83.6, 113.9, 114.8, 114.9,
133.4, 159.2 ppm. EI-MS: Calcd for C12H14O3 [M]+ 206.24, found: 206.09.
2-(9-bromo-6H-indolo[2,3-b]quinoxalin-6-yl)-N,N-dimethylethanamine (8)
1H NMR (300 MHz; CDCl3): δ 2.36 (s, 6H), 2.81 (t, 2H), 4.56 (t, J =
6.9 Hz, 2H), 7.37 (d, J = 8.4 Hz, 1H), 7.67–7.76 (m, 3H), 8.12 (d, J = 8.4 Hz,

Nucleic Acid 6H-Indolo[2,3-b] Quinoxaline Oligonucleotides
105
1H), 8.26 (d, J = 8.1 Hz, 1H), 8.57 (s, 1H) ppm. 13C NMR (75 MHz; CDCl3):
δ 40.0, 45.8, 57.2, 110.9, 113.6, 121.2, 125.4, 126.3, 127.9, 129.1, 129.4, 133.4,
139.5, 140.8, 142.9, 145.5 ppm. EI-MS: Calcd for C18H17BrN4 [M]+ 369.0709,
found: 369.0726.
(S)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[2,3-b]quinoxalin-9-yl)
ethynyl)phenoxy)butane-1,2-diol (9)
A
solution of 2-(9-bromo-6H-indolo[3,2-b]quinoxalin-6-yl)-N,N-
dimethylethan-amine 8 (0.23 g, 0.62 mmol), Pd(PPh3)2Cl2 (31 mg,
0.04 mmol), CuI (14 mg, 0.07 mmol), and powdered PPh3 (43 mg,
0.16 mmol) were dissolved in dry NEt3 (25 ml) and DMF (2 ml). The
reaction mixture was flushed with Ar for 30 minutes before (S)-4-(4-
ethynylphenoxy)butane-1,2-diol 7 (0.20 g, 0.93 mmol) was added. The
reaction mixture was stirred under Ar in a heavy walled smith process vial
at 120◦C for 25 minutes in the micro wave cavity. The reaction mixture was
washed with 0.3 M aq. EDTA (2 × 50 ml) to afford compound 9 as brown
1
solid without purification. Yield 0.33 g (89%). Mp 150–152◦C. H NMR
(300 MHz; DMSO-d6): δ 1.68 (m, 1H), 1.93 (m, 1H), 2.73 (s, 3H), 2.81 (t,
2H), 2.89 (s, 3H), 3.17 (m, 2H), 3.65 (m, 1H), 4.12 (m, 2H), 4.61(m, 2H),
7.01 (m, 2H), 7.54 (m, 2H), 7.76–7.89 (m, 3H), 7.95 (m, 1H), 8.12 (d, J =
8.4 Hz, 1H), 8.26 (d, J = 8.1 Hz, 1H), 8.47 (m, 1H) ppm. 13C NMR (75 MHz;
DMSO-d6): δ 32.9, 45.2, 56.6, 57.5, 64.7, 65.9, 67.9, 88.0, 88.7, 111.1,
114.6, 118.8, 124.6, 126.3, 127.6, 128.9, 131.5, 133.9, 134.3, 138.8, 140.0,
143.6, 145.3, 158.9 ppm. HRMS (MALDI) Calcd for C30H30N4O3 [M +
H]+ 495.2391, found: 495.2401.
(S)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-(4-((6-(2-(dimethylamino)
ethyl)-6H-indolo[2,3-b]quinoxalin-9-yl)ethynyl)phenoxy)butan-2-ol (10)
(S)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[3,2-b]quinoxalin-9-yl)
ethynyl)phenoxy)-butane-1,2-diol 9 (0.3 g, 0.61 mmol) was dissolved in dry
pyridine (20 ml) before 4,4ꢁ-dimethoxytrityl chloride (0.25 g, 0.73 mmol)
was added under N2. The reaction was stirred at room temperature
overnight; an extra portion of DMT-Cl (0.12 g, 0.37 mmol) was added
before it was quenched with MeOH (2 ml). The reaction mixture was
diluted with EtOAc (150 ml) before washing with saturated aq. NaHCO3
(2 × 40 ml). After back extraction with EtOAc (2 × 20 ml), the combined
organic phase was dried (Na2SO4), filtered, and concentrated under
reduced pressure. The residue was coevaporated with toluene–EtOH (2 ×
25 ml, 1: 1, v/v) before it was purified by column chromatography (silica
gel; 0.5% Et3N v/v, 30–70% EtOAc in cyclohexane) to afford 10 as a yellow
1
foam. Yield 0.25 g (52%). H NMR (300 MHz; DMSO-d6): δ 1.83 (m, 2H),
2.26 (s, 6H), 2.71 (m, 2H), 3.14 (m, 2H), 3.67 (s, 6H), 3.97 (m, 2H),
4.20 (m, 1H), 4.42 (m, 2H), 6.72 (d, J = 8.7 Hz, 5H), 7.16–7.25 (m, 7H),

106
A. M. A. Osman et al.
7.30–7.37 (m, 5H), 7.55–7.66 (m, 4H), 8.01 (d, J = 8.1 Hz, 1H), 8.15 (d, J =
8.1 Hz, 1H), 8.49 (m, 1H) ppm. 13C NMR (75 MHz; DMSO-d6): δ 33.1, 45.7,
46.0, 55.1, 57.0, 64.7, 67.4, 68.1, 86.0, 88.0, 88.7, 109.4, 113.0, 114.5, 116.1,
119.6, 125.8–130.8, 132.8, 133.9, 135.9, 139.4, 140.6, 143.4, 144.8, 145.7,
158.4, 158.7 ppm. HRMS (ESI) Calcd for C51H48N4O5 [M + H]+ 797.3697,
found: 797.3680.
(S)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-(4-((6-(2-(dimethylamino)
ethyl)-6H-Indolo[2,3-b]quinoxalin-9-yl)ethynyl)phenoxy)butan-2-yl
(2-cyanoethyl) diisopropylphosphoramidite (11)
(S)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-(4-((6-(2-(dimethyla-
mino)ethyl)-6H-indolo[2,3-b]quinoxalin-9-yl)ethynyl)phenoxy)butan-2-ol
10 (0.18 g, 0.23 mmol) and diisopropyl ammonium tetrazolide (0.06 g,
0.35 mmol) were dissolved under Ar in dry CH2Cl2 (20 ml), followed by drop
wise addition of 2-cyanoethyl N ,N ,N ꢁ,N ꢁ-tetraisopropylphosphordiamidite
(0.21 g, 0.69 mmol) via a syringe at 0◦C. The reaction mixture was stirred
under Ar at room temperature overnight before the reaction was quenched
with H2O (50 ml) and the organic phase was washed with H2O (2 × 50 ml).
After back extraction with CH2Cl2 (50 ml), the combined organic phase
was dried (MgSO4), filtrated, and concentrated under reduced pressure.
The residue was purified by dry column vacuum chromatography (silica
gel; Et3N 0.5% v/v, 50–50% EtOAc in cyclohexane) to afford 11 as a yellow
foam. Yield 0.2 g (87%). 1H NMR (300 MHz; CDCl3): δ 1.15–1.18 (m, 12H),
1.19 (m, 2H), 2.35 (s, 6H), 2.73 (t, J = 6.3 Hz, 2H), 2.81 (m, 2H), 3.44–3.55
(m, 6H), 3.78 (s, 6H), 4.09–4.17 (m, 3H), 4.53 (m, 2H), 6.84 (m, 5H),
7.19–7.51 (m, 12H), 7.62–7.79 (m, 4H), 8.15 (d, J = 8.1 Hz, 1H), 8.23 (d,
J = 8.1 Hz, 1H), 8.56 (m, 1H) ppm. 13C NMR (75 MHz; DMSO-d6): δ 20.0,
24.1, 24.2, 24.3, 24.4, 33.2, 42.7, 42.9, 44.9, 45.0, 54.9, 56.8, 57.8, 57.9, 63.9,
65.5, 70.6, 85.7, 87.8, 88.5, 109.3, 112.7, 114.3, 116.7, 119.3, 125.4–129.8,
132.5, 132.6, 135.8, 139.2, 140.3, 143.2, 144.7, 145.5, 158.1, 158.5 ppm. 31P
NMR (300 MHz; CDCl3): δ 149.53, 149.95 in a 4:3 ratio. HRMS (ESI) Calcd
for C60H65N6O6P [M +Na]+ 1019.4696, found: 1019.4669.
Oligonucleotide Synthesis, Purification, and Melting Temperature
Determination
DMT-on oligodeoxynucleotides were carried out at 0.2 μmol scales on
CPG supports with an ExpediteTM Nucleic Acid Synthesis System Model 8909
from (Applied Biosystems), using 1H- tetrazole as an activator for coupling
reaction and a 0.05 mM solution of the amidite (11) dissolved in anhydrous
CH2Cl2. And inserted into the growing oligonucleotides using a syringe at-
tached to the CPG-support and the solution was pressed through the support
giving a coupling time of 10 minutes. DMT-on oligonucleotides bound to
CPG supports were treated with aq. NH3 (32%, 1 cm3) at room temperature

Nucleic Acid 6H-Indolo[2,3-b] Quinoxaline Oligonucleotides
107
and then at 55◦C over night. Purification of 5ꢁ-O-DMT-on ONs was accom-
plished by reversed-phase semipreparative HPLC on a waters Xterra MS C18
column with a waters Delta Prep 4000 Preparative Chromatography System
{Buffer A [0.05M triethylammonium acetate in H2O (pH 7.4)] and Buffer B
(75% CH3CN in H2O)}. Flow 2.5 cm3/ minute. Gradients: 2 minutes 100%
A, linear gradient to 70% B in 38 minutes, Linear gradient to 100% B in
3 minutes, and then 100% A in 10 minutes). ODNs were DMT deprotected
in 100 μl 80% acetic acid for 20 minutes. afterwards, H2O (100 μl), and
aq. AcONa (3M, 50 μl) were added, and sodium per chlorate (15 μl) and
the ONs were precipitated from acetone (500 μl). All modified ODNs were
confirmed by MALDI-TOF analysis on a Voyager Elite Bio spectroscopy Re-
search Station from PerSeptive Biosystems. The purity of the final ODNs
was found to be over 90%, checked by ion-exchange chromatography using
LaChrom system from Merck Hitachi on Genpak-Fax column (Waters). Melt-
ing 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 concentration of 1.0 μM, followed by addition of the third
(TFO) strand at a concentration of 1.5 μM in a buffer consisting of sodium
cacodylate (20 mM), NaCl (100 mM), and MgCl2 (10 mM) at pH 6.0 or 7.2.
◦
ON2 m/z found: 4676.3; calcd: 4677.3. The melting temperature (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/minute). All melting temperatures are within the uncertainty 1.0◦C
as determined by repetitive experiments.
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