Intercalating nucleic acids (INAs) containing insertions of 6H-indolo[2,3-b]quinoxaline

Article abstract of DOI:10.1016/j.tet.2006.09.017

6H-Indolo[2,3-b]quinoxaline was studied as a covalently bound heteroaromatic intercalator. Six monomers were synthesized and incorporated into DNA oligonucleotides. Through a study of linker length dependence it was concluded that the linker between the o

Full text of DOI:10.1016/j.tet.2006.09.017

Tetrahedron 62 (2006) 11187–11199
Intercalating nucleic acids (INAs) containing insertions
of 6H-indolo[2,3-b]quinoxaline
y
*
Michael C. Wamberg, Allam A. Hassan, Andrew D. Bond and Erik B. Pedersen
Nucleic Acid Center,z Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Received 16 June 2006; revised 25 August 2006; accepted 8 September 2006
Available online 4 October 2006
Abstract—6H-Indolo[2,3-b]quinoxaline was studied as a covalently bound heteroaromatic intercalator. Six monomers were synthesized and
incorporated into DNA oligonucleotides. Through a study of linker length dependence it was concluded that the linker between the oligo and
the intercalator must consist of at least five C atoms in order to stabilize a DNA duplex. An intercalator with a 20-deoxy-D-riboside linker to the
oligo could also stabilize a DNA/RNA duplex, while (S)-4-(6-methylindolo[2,3-b]quinoxalin-3-ylmethoxy)-butane-1,2-diol was able to
stabilize both DNA/DNA, DNA/RNA and a DNA/LNA duplex. Mismatch studies revealed a huge sensitivity to the C–C mismatch at the
50-site of the intercalator.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
We have used the term intercalating nucleic acids to define
an oligonucleotide with an intercalating pseudonucleotide
inserted with a covalent bond. Important factors to be
considered while using intercalating nucleic acids for
hybridization are the structure of the backbone, and the
length of the linker and intercalator.14 We use a vicinal
dihydroxy system to incorporate the intercalator as a bulge
in the DNA backbone, because it creates a distance between
the intercalator and the neighbouring nucleobases of ca.
The term intercalation was first used by Lerman,1 who had
conducted a number of physical studies on interactions
of DNA with planar aromatic compounds, to describe the
non-covalent binding of a planar polyaromatic molecule
between the nucleobases of DNA. When intercalation
occurs, the torsion angles in the sugar-phosphodiester back-
bone of DNA are changed in order to accommodate the in-
tercalating aromatic moiety. This causes an unwinding of
˚
3.4 A. Furthermore it introduces additional flexibility into
˚
the DNA helix with a maximum helix increase of 3.4 A,
the backbone. Previously we have focused on pyrene as
the intercalating moiety and have achieved significant
discrimination between DNA and RNA,15 as the DNA
duplex was stabilized while the RNA duplex was destabi-
lized. The fluorescence properties of the pyrene intercalator
confirmed its intercalation rather than groove binding in
duplexes.16 These findings led to commercialization of
intercalating nucleic acids containing bulge insertions
of (R)-1-O-(1-pyrenylmethyl)glycerol (INAÔ) in order to
exploit the technology in diagnostics, e.g., DNA methylation
screening.17
which can be measured by viscosity and sedimentation of
the DNA solution.1 The degree of unwinding varies depend-
ing on the intercalator and the DNA sequence.2–4 Normally
˚
the helix increase is less than the maximal 3.4 A, because
of other effects such as bending of the duplex around the
site of intercalation. X-ray studies on intercalation were first
provided by Wang et al.5 Classical intercalators such as pro-
flavine, acridine, ethidium bromide, daunomycin6 and
actinomycin7,8 are known for their anti-tumour activity by
inhibition of topoisomerases,9,10 but the group of intercala-
tors has expanded heavily over the years and now includes
mono-, bis- and trisintercalators. For recent reviews, see
Martinez and Chacon-Garcia, Bran˜a et al.12 and Graves
For this study we were looking for a heteroaromatic inter-
calator in order to achieve higher affinity towards ssRNA.
Ren et al.18 had done some studies on the binding affinity
of small molecules to an RNA:DNA hybrid using a poly
rA:poly dTassay. Among the 84 tested compounds, of which
five compounds showed a potential, they found the best one
to be Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole
(1),19 a naturally occurring alkaloid, known for its promising
anti-tumour activities.20
11
ꢀ
and Velea.13
Keywords: Intercalating nucleic acids; 6H-Indolo[2,3-b]quinoxaline; Oligo-
nucleotides with a covalently bound intercalator; Thermal stability; DNA.
* Corresponding author. Tel.: +45 65502555; fax: +45 66158780; e-mail:
ebp@chem.sdu.dk
y
z
Present address: Faculty of Education, Suez Canal University, Egypt.
A research centre funded by The Danish National Research Foundation
for studies on nucleic acid chemical biology.
6H-Indolo[2,3-b]quinoxaline (2) can be seen as an aza
analogue of Ellipticine. It is a well-examined and
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.09.017

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M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
CH3
CH3
2. Results and discussion
N
H3C
N
N
2.1. Chemistry
N
N
CH3
The commercially available ((S)-(+)-2,2-dimethyl-1,3-di-
oxolane-4-yl)-methanol was mesylated in CH2Cl2/Et3N
according to the procedure described by Kim et al.31 to
give methanesulfonic acid (R)-2,2-dimethyl-1,3-dioxolane-
4-ylmethyl ester (10).32 Enantiomerically pure methane-
sulfonic acid 2-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-ethyl
ester (11)33 was obtained from (S)-malic acid in four steps.
(S)-Malic acid was converted to dimethyl (S)-malate accord-
ing to Mori and Ikunaka.34 The diester was reduced with
LiAlH4 to (S)-1,2,4-butanetriol and protected with an iso-
propylidene group as described by Hayashi et al.35 Mesyla-
tion of 2-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-ethanol with
methanesulfonyl chloride was carried out in either pyridine
according to Augustyns et al.33 or CH2Cl2/Et3N according
to Kim et al.31 Using pyridine as the solvent gave 11 in
51% yield, while the latter gave 11 in 99% yield. Enantio-
merically pure methanesulfonic acid 3-((S)-2,2-dimethyl-
1,3-dioxolan-4-yl)-propyl ester (12) was obtained from
L-glutamic acid in four steps. L-Glutamic acid was converted
to (S)-5-oxotetrahydrofuran-2-carboxylic acid as described
by Herdeis.36 Reduction with LiAlH4 according to Brunner
and Lautenschalger37 yielded (S)-1,2,5-pentanetriol. This
was protected with an isopropylidene group and mesylated
as described above to give 12.38
N
N
N
N(CH3)2
H
H
1
2
3
Figure 1. Structures of Ellipticine (1), 6H-Indolo[2,3-b]quinoxaline (2) and
B-220 (3).
well-described compound, primarily by Bergman and
co-workers.21–23 It has nitrogen atoms in the third ring
instead of the 5,11-methyl groups in Ellipticine. Several
analogues of 6H-indolo[2,3-b]quinoxaline have been syn-
thesized, e.g., B-220 (2,3-dimethyl-6-(2-dimethylamino-
ethyl)-6H-indolo[2,3-b]quinoxaline, 3), which have shown
high antiviral activity24,25 (Fig. 1).
Ellipticine (1) as well as B-220 (3) have been used as non-
covalent intercalators.21,22,26–29 Furthermore, Arimondo
et al.30 observed a stronger stabilization of a poly(dAdT)$
poly(dAdT) duplex for compound 2 than for its correspond-
ing pyridopyrazino[2,3-b]indole analogues. It was therefore
of interest to us to test 6H-indolo[2,3-b]quinoxaline as an
intercalating nucleic acid. We also chose indolo[2,3-b]-
quinoxaline as the intercalator for this study because of its
direct synthesis.
In this paper we present the synthesis of six intercalating
nucleic acids with indolo[2,3-b]quinoxaline as the intercala-
tor (Fig. 2) and their evaluation by thermal stability measure-
ments and fluorescence spectroscopy.
6H-Indolo[2,3-b]quinoxaline (2) and its 2,3-dichloro deriv-
ative (13) were easily prepared by condensation of isatin
with 1,2-phenylene diamine in glacial acetic acid according
to Schunck’s method.39,40 For the alkylation, 2 and 13 were
deprotonated (at N-6) with NaH giving red anions. Follow-
ing the procedure of Cassel et al.41 they were then reacted
with the mesylated alcohols 10 and 11 in refluxing DMF
for 48 h in the presence of the phase-transfer catalyst tetra-
n-butylammonium bromide (TBAB) giving 14b and c in
yields of 35–50%. A similar yield of 14d was achieved with-
out addition of TBAB,42 whereas this procedure afforded
14a in 76% yield using a long reaction time of three days.
For monomers 4–7 the covalently bound linker was attached
to N-6, which is the same position as the dimethylamino-
ethyl side chain that is attached in B-220 (3). Monomer 8
has a 20-deoxy-D-ribose as the connector to the backbone
of the DNA strand. It was synthesized in order to evaluate
the flexibility of the acyclic linkers versus the natural fura-
nose sugar linkage. The site of linker connection to the inter-
calator was changed in monomer 9 in order to investigate
whether this could lead to better base stacking as the interca-
lator would be positioned differently in the duplex.
The alkylation is supposed to take place on N-6 and this
molecule will be similar to B-220 (3). However, there is a
risk of forming an isomer due to alkylation on N-5. Bergman
and co-workers22,43 reported a 4:1 ratio of the 6-substituted
versus 5-substituted derivative, by using Knotz’s method.44
In this study we have investigated the dependence of linker
length on the stabilities of DNA/DNA, DNA/RNA and
DNA/LNA duplexes by varying the linker.
N
N
X
X
N
N
O
N
N
N
HO
N
N
n
HO
HO
9
O
HO
OH
4-7
OH
8
n
X
H
Cl
H
H
4
5
6
7
1
1
2
3
Figure 2.

M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
11189
b-isomer precipitated. Extraction with CHCl3 isolated the
a-isomer. Assignment of a,b-configuration was done by
2D NMR and NOE. Deprotection of the toluoyl groups
were carried out using sodium methoxide in methanol as
described by Abdel-Megied et al.46 yielding b-6-(20-deoxy-
ribose)-indolo[2,3-b]quinoxaline (8). DMT-protection of
the diol and conversion to the phosphoramidite (19) were
carried out as described above.
N
a
b
2
8
N
N
TolO
O
TolO
17
N
N
c
N
Figure 3. One independent molecule in the asymmetric unit of the X-ray
structure of 4. Displacement ellipsoids are shown at the 30% probability
level for non-H atoms.
DMTO
d
O
OR
18 R = H
19 R = P(NPri2)O(CH2)2CN
However, we did not isolate the N-5 alkylated isomer. The
N-6 alkylation was proven by obtaining a synchrotron
X-ray structure of the corresponding alcohol 4, a step further
in the synthetic route (see Figs. 2 and 3 and Scheme 1).
Scheme 2. (a) 2-Deoxy-3,5-di-O-(p-toluoyl)-a-D-pentofuranosyl chloride,
NaH, TBAB, DMF, 32%; (b) CH3ONa, MeOH, 73%; (c) DMT–Cl,
CH2Cl2/Et3N, 58%; (d) NC(CH2)2OP(NiPr2)2, N,N-diisopropylammonium
tetrazolide, CH2Cl2, 21%.
O
N
N
X
X
N
N
X
X
a
O
OMs
+
Having made 4–7 with three different linker lengths and
a sugar moiety (8), we also found it interesting to change
the connection site of the intercalator to the linker. This
was done in order to position the intercalator in a different
way inside the duplex and thereby to investigate, whether
better base stacking could be obtained. At the same time
the linker length was further increased.
n
N
N
H
10 n = 1
11 n = 2
12 n = 3
n
2 R = H
13 R = Cl
O
O
14a-d
X
N
N
b
c
4-7
N
X
6H-Indolo[2,3-b]quinoxaline-3-carboxylic acid (20) was
synthesized by condensation of isatin with 3,4-diaminobenz-
oic acid in glacial acetic acid according to Schunck’s
method.39,40 As the diamino compound is not symmetric,
there was a possibility of regioisomers. However, we did
not isolate the regioisomeric 6H-indolo[2,3-b]quinoxaline-
2-carboxylic acid. The regioselectivity has previously been
reported by Varma and Khan.47 However, we found it
more convincing to prove the structure 20 by an X-ray struc-
ture of the corresponding alcohol 23, a few steps further in
the synthetic route (see Fig. 4 and Scheme 3).
n
14-16
n
X
ODMT
RO
H
Cl
H
H
a
b
c
d
1
1
2
3
15a-d R = H
16a-d R = P(NPri2)O(CH2)2CN
d
Scheme 1. (a) NaH, TBAB, DMF, 35–76%; (b) AcOH/H2O (4:1), 67–93%;
(c) DMT–Cl, pyr or CH2Cl2/Et3N, 24–83%; (d) NC(CH2)2OP(NiPr2)2,
N,N-diisopropylammonium tetrazolide, CH2Cl2, 61–84%.
Treatment of 14a–d with 80% aq acetic acid gave the diols
4–7. These were DMT-protected using DMT–chloride in
pyridine or in CH2Cl2/Et3N and then converted to the phos-
phoramidites 16a–d using 2-cyanoethyl-N,N,N0,N0-tetra-
isopropylphosphane in CH2Cl2.
In order to avoid alkylation on N-6, when alkylating the new
site (at the 3 position), the N-6 position was blocked with
a methyl group using CH3I in DMSO in the presence of pow-
dered potassium hydroxide. This yielded a 3:2 mixture of
6-methylindolo[2,3-b]quinoxaline-3-carboxylic acid methyl
ester (21) and the carboxylic acid (22). A small sample of the
crude product was separated into pure compounds. The rest
was reduced to the corresponding alcohol (6-methyl-
indolo[2,3-b]quinoxalin-3-yl)methanol (23) using LiAlH4
in THF. Badger and Nelson48 reported a 40% yield of the
6-methylated compound and 12% of the 5-methylated by
We also synthesized an analogue having a 20-deoxy-D-ribo-
side as the backbone linker (Scheme 2). 2-Deoxy-3,5-
di-O-(p-toluoyl)-a-D-pentofuranosyl chloride was prepared
according to Rolland et al.45 and coupled with 6H-indolo-
[2,3-b]quinoxaline (2) using NaH in DMF according to
the procedure described above.41 a- and b-Isomers were
separated by dissolving the mixture in MeCN by which the

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M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
In order to have a more reactive leaving group for the sub-
sequent alkylation reaction, the alcohol 23 was converted
to 3-chloromethyl-6-methylindolo[2,3-b]quinoxaline (24)
using thionyl chloride in CH2Cl2 according to the procedure
described by Bair et al.49 Following the procedure of Tirosh
et al.,50 24 was reacted with 2-((S)-2,2-dimethyl-1,3-dioxo-
lan-4-yl)-ethanol in toluene in the presence of powdered
potassium hydroxide to give 25. Subsequent treatment of
25 with 80% acetic acid as described above, followed by
DMT-protection and phosphitylation gave 27 (Scheme 3).
The DMT-protected phosphoramidites of the intercalating
nucleic acid monomers 4–9 were incorporated into two dif-
ferent DNA oligonucleotides using the same coupling times
(2 min) in the oligo synthesis as was used for the amidites of
natural nucleosides. The monomers 4–9 were inserted as
a bulge in a 12-mer highly conserved HIV-1 strand,51 which
was modified around the site of intercalation (C-G switch)
according to Christensen et al.14,15 They were also inserted
as a bulge or as an end-positioned intercalating pseudo-
nucleotide in an 11-mer oligonucleotide earlier described
by Filichev et al.52 All modified oligonucleotides were con-
firmed by MALDI-TOF analysis with a variation of m/z ꢀ3.
Figure 4. X-ray structure of 23$H2O showing displacement ellipsoids at the
50% probability level for non-H atoms.
N
N
a
N
H
N
HOOC
ROOC
N
N
20
21 R = CH3 + 22 R = H (3:2)
2.2. Thermal melting studies
N
N
b
c
HO
Cl
N
N
N
24
The project was designed to study the linker length depen-
dence of the intercalating nucleic acids. As it is seen from
Table 1, intercalators with short linkers (4–6) were destabi-
lizing the DNA duplex, whereas stabilization was obtained
when a longer linker (7) or a sugar moiety (8) is used. Intro-
duction of chloro substituents in the 2 and 3 positions of the
6H-indolo[2,3-b]quinoxaline (5) gave a marginal improve-
ment compared to 4. Highest stabilization was obtained for
9, which had the longest linker, and the linker attached to
C-3 instead of N-6. It is believed that the shorter linker is
unable to position the intercalator optimally for base stack-
ing in the duplex without disturbance of the backbone.
N
23
N
e
d
f
9
O
N
N
25
O
O
N
O
N
N
DMTO
OR
26 R = H
27 R = P(NPri2)O(CH2)2CN
In comparison with previous studies14 of pyrene and anthra-
cene intercalators inserted in the same sequence and with the
same linker length, 6H-indolo[2,3-b]quinoxaline (9) gives
a higher DTm (+4.5 ꢁC vs +2.5 ꢁC for pyrene and ꢂ1.3 ꢁC
for anthracene).
g
Scheme 3. (a) CH3I, KOH, DMSO; (b) LiAlH4, THF, 44% (over two steps);
(c) SOCl2, CH2Cl2, 89%; (d) (S)-2,2-dimethyl-1,3-dioxolane-4-ethanol,
KOH, toluene, 67%; (e) AcOH/H2O (4:1); (f) DMT–Cl, CH2Cl2/Et3N,
55%; (g) NC(CH2)2OP(NiPr2)2, N,N-diisopropylammonium tetrazolide,
CH2Cl2, 47%.
Shifting the intercalator to the complementary strand (Table
1, bottom) was expected to give higher Tm as better stacking
is normally obtained when the intercalator is neighbouring
two guanines in the same strand rather than two cytosines.
This was also observed with the linkers 4–7. However,
the opposite effect is the case, when the acyclic linker
is replaced with the 2-deoxyribose (8) or the elongated
using 1 equiv of KOH and CH3I in ethanol. We used an
excess of both KOH and CH3I and DMSO as solvent, which
according to Zegar et al.22 should give a 95:5 ratio, but we
isolated only the 6-methylated compound, which was also
proven by the X-ray structure of 23$H2O (Fig. 4).
Table 1. Melting temperatures of duplexes with 4, 5, 6, 7, 8 and 9 inserted as a bulge
X¼
Sequences
—
4
5
6
7
8
9
Tm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
ODN1
ODN2
50-AGCTTG–GTTGAG-30
30-TCGAACXCAACTC-50
49.6
49.6
ꢂ9.0
ꢂ6.0
ꢂ8.3
ꢂ5.7
ꢂ1.0
ꢂ1.9
+1.7
+4.3
+4.0
+2.2
+4.5
ODN3
ODN4
50-AGCTTGXGTTGAG-30
30-TCGAAC–CAACTC-50
ꢂ0.9
DTm is the difference in Tm between duplexes with an intercalator (X) inserted and the corresponding unmodified duplex.

M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
11191
6
5
4
3
2
1
0
resulted in destabilization. This has also been the case in
other studies with INA54 with two pyrenes opposing each
other in a slightly different DNA sequence. The stabilization
of 8 is most likely due to zipping of the two indolo[2,3-b]qui-
noxaline intercalating moieties. Also 9 was stabilizing the
duplex when two intercalating moieties were inserted oppo-
site to each other. In this case zipping may be facilitated by
the longer linker.
ODN1 + ODN2
ODN3 + ODN4
ss-ODN3
ss-ODN2
For further investigation on indolo[2,3-b]quinoxaline as
an intercalator, monomers 4–9 were incorporated into a
11-mer oligonucleotide52 either in a region of A$T base pairs
in the middle of the strand or as an end positioned pseudo-
nucleotide. Thermal stability measurements were made
against DNA, RNA and LNA55 strands (Table 3).
350
400
450
500 550
wavelength
600
650
700
Figure 5. Fluorescence spectra of 9 inserted in ODN2 and ODN3.
linker/intercalator system 9. For 9, a destabilization of the
duplex was even observed contrary to what is seen, when the
linker is connected to N-6 and when pyrene is used as the in-
tercalator.14 For the same linker length, shifting the pyrene
intercalator to the complementary strand resulted in further
stabilization of the duplex (DTm¼3.6 ꢁC).14 With the linker
attached to C-3 of indolo[2,3-b]quinoxaline, the fourth ring
of the intercalator can reach deeply into the duplex, which is
not the case with pyrene because of its shape. It is thus antici-
pated that the intercalator 9 is placed nearer to the nucleo-
bases of the complementary strand than those of its ‘own’
strand and in this way it is capable of stacking to guanines
in the opposite strand in a better way.
Also for this sequence the dependence on linker length was
obvious. When incorporated in the middle of the sequence,
4, 5 and 6 destabilized the DNA/DNA duplex. A linker
with n¼3 (7) was necessary in order to achieve stabilization.
The RNA/DNA duplex stabilization was only observed for
the deoxyribose linked intercalators 8 and 9 with the longest
linker attached to C-3.
Incorporation of 50-end pseudonucleotide gave stabilization
to all the duplexes and for all the investigated linkers. It is
seen that the chloro substituents do not lead to further stabi-
lization of the duplex when compared with 4. However, it is
noteworthy that 8, which gives the highest Tm when incorpo-
rated into the middle of the DNA duplex, gives a lower Tm
than 7 and 9 when incorporated at the 50-end of a duplex.
This might be due to the greater flexibility of the acyclic
linker.
This hypothesis is supported by fluorescence spectroscopy
(Fig. 5). Weak fluorescence was observed for the ss-DNA
containing insertions of 9 between two guanines. When 9
was inserted between two cytosines in the ss-DNA, the fluo-
rescence was stronger. Upon hybridization to the comple-
mentary strand, the monomer fluorescence of 9 inserted
between the two cytosines was quenched. To the contrary,
enhancement of fluorescence was observed when the
ss-DNA containing insertions of 9 between two guanines
was hybridized to the complementary strand. This implies
that the intercalator in the first case makes base stacking
with the neighbouring guanines in the opposite strand, which
also leads to stabilization of the duplex.
For the affinity studies towards LNA, we did not use a fully
modified duplex but incorporated three thymines with LNA
(TL) into a DNA duplex. LNA locks the sugar ring in a north-
type conformation, resulting in an A-type duplex, which is
typically seen for RNA/RNA duplexes. If the duplex is not
fully modified with LNA monomers, there will only be
A-type duplex around the insertions. In the rest of the
duplex—away from LNA monomers—there will be the
B-type structure like in DNA/DNA duplexes. However, in
this case, with three LNA insertions evenly distributed, it
is reasonable to anticipate an A-type structure of the duplex
as revealed by NMR structure determination of a similar
LNA/DNA duplex.56,57 Except for 9 all intercalating nucleic
acid monomers destabilized the DNA/LNA duplex when
they were inserted as a bulge in the middle of the sequence.
It is interesting to observe the discrimination of 7 and 8
among DNA/LNA, DNA/RNA and DNA/DNA duplexes.
Compound 7 stabilized the DNA/DNA duplex, but destabi-
lized the DNA/LNA and DNA/RNA duplexes, even though
the latter was only marginally destabilized (DTm¼ꢂ0.7 ꢁC).
The intercalators were also placed opposite to each other in
the two strands of the duplex in a zipping manner (Table 2).
We observed a significant difference between 4 and 5 as the
2,3-dichloro substituted 5 was destabilizing the duplex by
ꢂ4.4 ꢁC, while 4 gave a destabilization of ꢂ11.5 ꢁC. Com-
pound 7 was marginally stabilizing the duplex. The most
important result was obtained with the 20-deoxyribose link-
age 8, which gave a significant stabilization. This result is
remarkable, because previous studies53 on two pyrenes
directly opposing each other in the same duplex sequence
Table 2. Melting temperatures of duplexes with zipping insertions of 4, 5, 7, 8 and 9
X¼
Sequences
—
4
5
7
8
9
Tm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
ODN3
ODN2
50-AGCTTGXGTTGAG-30
30-TCGAACXCAACTC-50
49.6
ꢂ11.5
DTm is the difference in Tm between duplexes with an intercalator (X) inserted and the corresponding unmodified duplex.
ꢂ4.4
+0.3
+9.7
+6.6

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M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
Table 3. Comparison of DNA/DNA, DNA/RNA and DNA/LNA duplexes with 4, 5, 7, 8 and 9 inserted as a bulge or at the end of the duplex
X¼
Sequences
—
4
5
6
7
8
9
Tm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
DTm (ꢁC)
50-TGTGAT–ATGCT-30
30-ACACTAXTACGA-50
42.4
42.4
ꢂ9.9
ꢂ6.8
ꢂ1.9
2.4
3.8
4.6
3.4
3.4
4.6
50-TGTGATATGCT-30
30-ACACTATACGAX-50
2.4
2.4
—
50-UGUGAU–AUGCU-30
30-ACACTAXTACGA-50
39.5
39.5
ꢂ13.4
ꢂ11.8
—
—
ꢂ0.7
1.8
1.5
2.3
1.6
50-UGUGAUAUGCU-30
30-ACACTATACGAX-50
0.7
0.4
1.7
50-TGTLGATLꢂATLGCT-30
30-ACA CTA XTA CGA-50
56.7
56.7
ꢂ15.6
ꢂ16.9
ꢂ9.9
ꢂ4.4
ꢂ4.6
1.6
4.6
50-TGTLGATL–ATLGCT-30
30-ACA CTA TAC GAX-50
2.1
2.9
—
3.5
3.1
T
L denotes locked nucleotide of thymine. DTm is the difference in Tm between duplexes with an intercalator (X) inserted and the corresponding unmodified
duplex.
Table 4. Melting temperatures of mismatched sequences with 4, 7, 8 and 9 inserted as a bulge
Sequence: 50-AGC TTZ YTT GAG-30
30-TCG AAC X CAACTC-50
X¼
—
4
7
8
9
Z
Y
Tm (ꢁC)
DTm (ꢁC)
Tm (ꢁC)
DTm (ꢁC)
Tm (ꢁC)
DTm (ꢁC)
Tm (ꢁC)
DTm (ꢁC)
Tm (ꢁC)
DTm (ꢁC)
Wild type
Mut. 1
Mut. 2
Mut. 3
Mut. 4
Mut. 5
Mut. 6
G
G
G
G
C
A
T
G
C
A
T
G
G
G
49.6
21.7
33.6
32.4
28.2
31.1
32.2
40.6
17.9
28.3
28.4
30.3
30.5
30.5
51.3
20.4
31.6
30.5
35.3
36.4
37.2
53.6
22.3
34.7
33.9
37.3
38.4
39.7
54.1
37.1
38.5
40.2
37.2
38.4
38.1
ꢂ27.9
ꢂ16.0
ꢂ17.2
ꢂ21.4
ꢂ18.5
ꢂ17.4
ꢂ22.7
ꢂ12.3
ꢂ12.2
ꢂ10.3
ꢂ10.1
ꢂ10.1
ꢂ30.9
ꢂ19.7
ꢂ20.8
ꢂ16.0
ꢂ14.9
ꢂ14.1
ꢂ31.3
ꢂ18.9
ꢂ19.7
ꢂ16.3
ꢂ15.2
ꢂ13.9
ꢂ17.0
ꢂ15.6
ꢂ13.9
ꢂ16.9
ꢂ15.7
ꢂ16.0
DTm is the difference in Tm between the matched sequence and the mismatched.
Compound 8 stabilized both DNA/DNA and DNA/RNA du-
plexes, but destabilized the DNA/LNA duplex. Compound 9,
which was stabilizing the investigated DNA/DNA, DNA/
RNA and DNA/LNA duplexes, is currently being examined
for stabilization of Three Way Junctions (TWJ).
not increase stabilization significantly and therefore 2,3-di-
chloro-6H-indolo[2,3-b]quinoxaline was not coupled to
any of the longer linkers.
In comparison studies of a DNA/DNA, DNA/RNA and
a DNA/LNA duplex the linker length dependence was also
obvious. When inserted in an A$T region in the middle of
a 11-mer sequence, only 9 could stabilize DNA/LNA, while
8 and 9 stabilized DNA/RNA. Stabilization of a DNA/DNA
duplex could be achieved using 7, 8 or 9.
Finally mismatch studies were carried out (Table 4). The
specificity for hybridization was measured by the difference
in the melting temperature between the fully complementary
duplex and the duplex where one mismatch has been intro-
duced. In general, when a mismatch was introduced at the
30-site of the intercalator, this proved to be less sensitive
than the unmodified oligo, while a greater sensitivity was
observed when the mismatch was introduced at the 50-site
of the intercalator. However, for the C–C mismatch at the
50-site of the intercalator, monomers 7 and 8 caused a drop
in melting temperatures up to ca. 30 ꢁC.
In mismatch studies we found the intercalator to be less
sensitive to mismatch at the 30-site than observed for the
unmodified duplex and more sensitive to the 50-site of the
intercalator. With insertions of the 6H-indolo[2,3-b]qui-
noxaline containing monomers 7 and 8 the maximum drop
in melting temperature was ca. 30 ꢁC for a C–C mismatch.
3. Conclusion
4. Experimental
4.1. General
From the study of linker length dependence we conclude that
the linker must, as a minimum, have a length corresponding
to 7, when used as a covalently bound intercalator. Shorter
linkers destabilize the duplexes as they are not able to posi-
tion the intercalator optimally for base stacking without
disturbing the backbone. Introduction of chloro substituents
in the 2,3 positions of 6H-indolo[2,3-b]quinoxaline did
NMR spectra were recorded on a Varian Gemini 2000 NMR
spectrometer at 300 MHz for 1H, 75 MHz for 13C and
121.5 MHz for 31P with TMS as an internal standard for
1H NMR, deuterated solvents CDCl3 (d 77.00), CD2Cl2 (d
53.80), CD3OD (d 49.00), DMSO (d 39.44) for 13C NMR,

M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
11193
and 85% H3PO4 as an external standard for 31P NMR.
MALDI mass spectra were recorded on a Fourier Transform
Ion Cyclotron Resonance Mass Spectrometer (Ionspec,
Irvine, CA). For accurate ion mass determinations, the
[M+H]+ or [M+Na]+ ion was peak matched using ions
derived from the 2,5-dihydroxybenzoic acid matrix. IR spec-
tra were recorded on a Perkin–Elmer 1720 Infrared Fourier
Transform Spectrometer. Melting points were determined
NMR (CDCl3): d 25.57, 27.05 (2ꢃCH3), 32.60 (C-20),
38.36 (C-10), 69.18 (C-40), 73.47 (C-30), 109.10 (C(CH3)2),
109.54, 119.42, 120.91, 122.66, 125.97, 127.75, 128.71,
129.32, 130.94, 139.29, 140.08, 140.52, 144.45, 145.47
(Carom). HRMS (MALDI): m/z calcd for C21H22N3O2+
(MH+): 348.1706, found 348.1705.
4.3. General procedure for alkylation of 2 and 13,
method B42
€
on a Buchi melting point apparatus. Silica gel (0.040–
0.063 mm) used for column chromatography and analytical
silica gel TLC plates 60 F254 were purchased from Merck.
UV-light or a stain of (NH4)6Mo7O24$4H2O/Ce2(SO4)3
(50:1) in 5% sulfuric acid was used for visualization. Sol-
vents used for column chromatography were distilled prior
to use, while reagents were used as purchased. Petroleum
ether (PE): bp 60–80 ꢁC. NMR assignment follows standard
nucleoside style, that is the carbon next to the intercalator is
assigned C-10.
NaH (60% in oil, 1.2 equiv) was added in two portions to
a solution of 6H-indolo-[2,3-b]quinoxaline39,40 (2, 0.230 g,
1 mmol) in dry DMF (20 mL). The reaction mixture was
heated to 80 ꢁC for 30 min, before the mesylated alcohol
10 (0.405 g, 2 mmol, 2 equiv) or 12 (0.250 g, 1 mmol,
1 equiv) was added dropwise. The reaction mixture was
stirred at 80 ꢁC overnight (14d) or for three days (14a).
The mixture was partitioned between CH2Cl2 and brine.
The organic phase was dried (MgSO4), and the solvent evap-
orated by co-evaporation with dry xylene. The product was
purified by silica gel column chromatography using
EtOAc/PE (25:75 v/v) as an eluent.
4.2. General procedure for alkylation of 2 and 13,
method A41
NaH (60% in oil, 1.5 equiv) was added portionwise to a
solution of 6H-indolo[2,3-b]quinoxaline39,40 (2, 0.139 g,
0.63 mmol) or 2,3-dichloro-6H-indolo-[2,3-b]quinoxaline58
(13, 0.250 g, 0.87 mmol) in dry DMF (40 mL). After stirring
at rt for 30 min, TBAB (0.2 equiv) was added and the
reaction mixture was stirred for additional 30 min. The
mesylated alcohol 10 or 11 (2 equiv) was added dropwise.
The reaction mixture was stirred at 140 ꢁC for 48 h. After
cooling, DMF was evaporated off under reduced pressure.
The residue was treated with water and extracted three times
with CHCl3. The combined organic phases were dried
(MgSO4) and the solvent evaporated off. The product was
purified by silica gel column chromatography using
EtOAc/PE (25:75 v/v) as an eluent.
4.3.1. 6-((S)-2,2-Dimethyl-1,3-dioxolan-4-ylmethyl)-6H-
indolo[2,3-b]quinoxaline (14a). Yield: 0.268 g (76%) as
1
a yellow solid, Rf 0.44 (EtOAc/PE 1:1), mp 135 ꢁC. H
NMR (CDCl3): d 1.33 (s, 3H, CH3), 1.39 (s, 3H, CH3),
4.00 (dd, 1H, J¼5.5 Hz, 8.6 Hz, H-30), 4.13 (dd, 1H,
J¼6.0 Hz, 8.6 Hz, H-30), 4.54 (dd, 1H, J¼5.5 Hz, 14.4 Hz,
H-10), 4.65 (dd, 1H, J¼5.5 Hz, 14.4 Hz, H-10), 4.70 (quintet,
1H, J¼5.5 Hz, H-20), 7.38 (m, 1H, Harom), 7.62–7.78 (m, 4H,
Harom), 8.11 (dd, 1H, J¼1.5 Hz, 8.3 Hz, Harom), 8.31 (dd,
1H, J¼1.5 Hz, 8.3 Hz, Harom), 8.46 (dd, 1H, J¼0.7 Hz,
7.8 Hz, Harom). 13C NMR (CDCl3): d 25.20, 26.68
(2ꢃCH3), 44.31 (C-10), 67.32 (C-30), 74.49 (C-20), 109.76
(C(CH3)2), 110.41, 119.47, 121.14, 122.53, 126.13,
127.75, 128.50, 129.25, 130.90, 139.40, 139.97, 140.43,
144.89, 145.80 (Carom). HRMS (MALDI): m/z calcd for
C20H19N3O2Na+ (MNa+): 356.1369, found 356.1377.
4.2.1. 2,3-Dichloro-6-((S)-2,2-dimethyl-1,3-dioxolan-
4-ylmethyl)-6H-indolo[2,3-b]quinoxaline (14b). Yield:
0.122 g (35%) as a yellow solid, Rf 0.94 (EtOAc/PE 2:3),
mp 131 ꢁC. 1H NMR (CDCl3): d 1.32, 1.38 (2ꢃs, 6H,
2ꢃCH3), 3.97 (dd, 1H, J¼5.5 Hz, 8.6 Hz, H-30), 4.15 (dd,
1H, J¼5.5 Hz, 8.6 Hz, H-30), 4.52 (dd, 1H, J¼5.5 Hz,
14.6 Hz, H-10), 4.61 (dd, 1H, J¼5.5 Hz, 14.6 Hz, H-10),
4.69 (quintet, 1H, J¼5.5 Hz, H-20), 7.40 (t, 1H, J¼7.4 Hz,
Harom), 7.61–7.74 (m, 2H, Harom), 8.19 (s, 1H, Harom), 8.36
(s, 1H, Harom), 8.39 (d, 1H, J¼7.9 Hz, Harom). 13C NMR
(CDCl3): d 25.17, 26.70 (2ꢃCH3), 44.49 (C-10), 67.24
(C-30), 74.36 (C-20), 109.10 (C(CH3)2), 110.70, 119.15,
121.64, 122.81, 128.29, 131.62, 132.87, 138.10, 139.18,
145.23 (Carom). HRMS (MALDI): m/z calcd for
C20H18Cl2N3O+2 (MH+): 402.0770, found 402.0783.
4.3.2. 6-[3-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)propyl]-
6H-indolo[2,3-b]quinoxaline (14d). Yield: 0.132 g (35%)
1
as a yellow oil. H NMR (CDCl3): d 1.34 (s, 3H, CH3),
1.38 (s, 3H, CH3), 1.63–1.73 (m, 2H, H-30), 2.02–2.10 (m,
2H, H-20), 3.47 (t, 1H, J¼8.0 Hz, H-50), 3.99 (dd, 1H, J¼
5.9 Hz, 8.0 Hz, H-50), 4.14–4.19 (quintet, 1H, J¼5.9 Hz,
H-40), 4.52 (dd, 2H, J¼6.7 Hz, 13.4 Hz, H-10), 7.38 (t, 1H,
J¼07.6 Hz, Harom), 7.47 (d, 1H, J¼8.3 Hz, Harom), 7.64–
7.77 (m, 3H, Harom), 8.12 (dd, 1H, J¼1.5 Hz, 8.3 Hz, Harom),
8.29 (dd, 1H, J¼1.5 Hz, 8.3 Hz, Harom), 8.47 (d, 1H, J¼
13
7.6 Hz, Harom). C NMR (CDCl3): d 24.83 (C-20), 25.64,
26.90 (2ꢃCH3), 30.66 (C-30), 41.12 (C-10), 69.23 (C-50),
75.50 (C-40), 108.84 (C(CH3)2), 109.41, 119.46, 120.82,
122.72, 125.91, 127.75, 128.68, 129.29, 130.90, 139.25,
139.93, 140.54, 144.24, 145.58 (Carom). HRMS (MALDI):
m/z calcd for C22H23N3O2Na+ (MNa+): 384.1682, found
384.1691.
4.2.2. 6-[2-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)ethyl]-
6H-indolo[2,3-b]quinoxaline (14c). Yield: 0.110 g (50%)
as a yellow oil, Rf 0.76 (EtOAc/PE 1:4). 1H NMR
(CDCl3): d 1.32 (s, 3H, CH3), 1.47 (s, 3H, CH3), 2.13–
2.25 (m, 2H, H-20), 3.57 (dd, 1H, J¼7.1 Hz, 8.0 Hz, H-40),
4.01 (dd, 1H, J¼6.0 Hz, 8.0 Hz, H-40), 4.11–4.19 (m, 1H,
H-30), 4.52–4.70 (m, 2H, H-10), 7.37 (m, 1H, Harom), 7.56
(d, 1H, J¼8.2 Hz, Harom), 7.64–7.77 (m, 3H, Harom), 8.11
(dd, 1H, J¼1.2 Hz, 8.2 Hz, Harom), 8.29 (dd, 1H, J¼
1.2 Hz, 8.2 Hz, Harom), 8.46 (d, 1H, J¼7.2 Hz, Harom). 13C
4.4. General procedure for isopropylidene deprotection
Compounds 14a–d were stirred in acetic acid/H2O (4:1,
50 mL) at rt overnight. The solvent was evaporated under
reduced pressure.

11194
M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
4.4.1. (S)-3-(Indolo[2,3-b]quinoxalin-6-yl)-propane-1,2-
diol59 (4). Yield: 0.194 g (93%) as yellow crystals (obtained
from 0.237 g, 0.71 mmol of 14a), Rf 0.32 (4% MeOH/
for 30 min, TBAB (0.30 g, 0.9 mmol, 0.2 equiv) was added
and the reaction mixture was stirred for another 30 min.
2-Deoxy-3,5-di-O-(p-toluoyl)-a-D-pentofuranosyl chloride
(1.64 g, 4.2 mmol, 0.9 equiv) was added dropwise. The reac-
tion mixture was stirred at rt for 48 h. The solvent was evap-
orated off under reduced pressure, and the residue was
purified by silica gel column chromatography using
EtOAc/PE (40:60 v/v) as an eluent. a- and b-Isomers were
separated by dissolving the mixture in MeCN by which the
b-isomer precipitated. Extraction with CHCl3 isolated the
a-isomer.
CH2Cl2), mp 215 ꢁC. IR (KBr): 3338 cmꢂ1 1H NMR
.
(DMSO-d6): d 3.52 (d, 2H, J¼5.3 Hz, H-30), 4.14 (m, 1H,
H-20), 4.45 (dd, 1H, J¼7.8 Hz, 14.4 Hz, H-10), 4.56 (dd,
1H, J¼4.4 Hz, 14.4 Hz, H-10), 4.89 (br s, 1H, OH), 5.03
(br s, 1H, OH), 7.41 (t, 1H, J¼7.7 Hz, Harom), 7.71–7.86
(m, 4H, Harom), 8.12 (dd, 1H, J¼8.3 Hz, 1.1 Hz, Harom),
8.28 (dd, 1H, J¼8.3 Hz, 1.1 Hz, Harom), 8.38 (d, 1H,
13
J¼7.7 Hz, Harom). C NMR (DMSO-d6): d 44.93 (C-10),
63.96 (C-30), 69.60 (C-20), 111.14, 118.54, 120.77,
121.91, 125.94, 127.50, 128.88, 129.04, 131.11, 138.58,
139.63, 139.89, 145.09, 145.44 (Carom). HRMS (MALDI):
m/z calcd for C17H15N3O2Na+ (MNa+): 316.1056, found
316.1054.
1
Yield: 0.77 g (32%) as a yellow solid. H NMR (CDCl3):
d 2.40, 2.46 (2ꢃs, 6H, 2ꢃCH3), 2.65 (ddd, 1H, J¼
14.3 Hz, 6.6 Hz, 2.9 Hz, H-20), 3.76 (dt, J¼14.3 Hz,
6.6 Hz, H-200), 4.65 (q, 1H, J¼3.7 Hz, H-40), 4.74 (dd, 1H,
J¼11.9 Hz, 4.3 Hz, H-50), 4.94 (dd, 1H, J¼11.9 Hz,
3.7 Hz, H-50), 6.08 (dt, 1H, J¼7.4 Hz, 6.6 Hz, H-30), 7.10
(t, 1H, J¼6.6 Hz, H-10), 7.17 (d, 2H, J¼8.0 Hz, Harom),
7.31 (d, 2H, J¼8.0 Hz, Harom), 7.35–7.42 (m, 2H, Harom),
7.68–7.80 (m, 3H, Harom), 7.94 (d, 2H, J¼8.0 Hz, Harom),
8.05 (d, 2H, J¼8.0 Hz, Harom), 8.16 (dd, 1H, J¼8.1 Hz,
1.5 Hz, Harom), 8.29 (dd, 1H, J¼8.1 Hz, 1.5 Hz, Harom),
8.46 (dd, 1H, J¼8.1 Hz, 1.5 Hz, Harom). 13C NMR
(CDCl3): d 21.68, 21.74 (2ꢃCH3), 34.73 (C-20), 63.95
(C-50), 74.64 (C-30), 81.42 (C-40), 83.43 (C-10), 111.61,
120.33, 121.83, 122.72, 126.66, 126.73, 126.92, 128.05,
129.10, 129.12, 129.26, 129.69, 129.84, 130.94, 139.61,
140.02, 142.85, 143.87, 144.31, 145.18 (Carom), 166.15,
166.29 (2ꢃC]O). HRMS (MALDI): m/z calcd for
C35H29N3O5Na+ (MNa+): 594.1999, found 594.2005.
4.4.2. (S)-3-(2,3-Dichloro-indolo[2,3-b]quinoxalin-6-yl)-
propane-1,2-diol (5). Yield: 0.080 g (92%) as a yellow solid
(obtained from 0.097 g, 0.24 mmol of 14b), Rf 0.50 (5%
1
MeOH/CHCl3), mp 212 ꢁC. H NMR (DMSO-d6): d 3.50
(t, 2H, J¼5.4 Hz, H-30), 4.07–4.12 (m, 1H, H-20), 4.41 (dd,
1H, J¼7.9 Hz, 14.2 Hz, H-10), 4.51 (dd, 1H, J¼4.2 Hz,
14.2 Hz, H-10), 4.83 (t, 1H, J¼5.5 Hz, OH), 4.97 (d, 1H, J¼
5.3 Hz, OH), 7.42 (m, 1H, Harom), 7.80 (m, 3H, Harom), 8.33–
8.36 (m, 2H, Harom). 13C NMR (DMSO-d6): d 44.49 (C-10),
63.91 (C-30), 69.44 (C-20), 111.43, 118.01, 121.28, 122.28,
128.10, 129.58, 131.92, 137.13, 138.85, 145.19 (Carom).
HRMS (MALDI): m/z calcd for C17H14Cl2N3O+2 (MH+):
362.0457, found 362.0472.
4.4.3. (S)-4-(Indolo[2,3-b]quinoxalin-6-yl)-butane-1,2-
diol (6). Yield: 0.065 g (67%) as a yellow oil (obtained
from 0.110 g, 0.32 mmol of 14c), Rf 0.20 (EtOAc/PE 1:4).
1H NMR (DMSO-d6): d 1.74–1.87 (m, 1H, H-20), 2.07–
2.18 (m, 1H, H-20), 3.26–3.57 (m, 3H, H-30, H-40), 4.50–
4.67 (m, 2H, H-10), 4.83 (br s, 2H, 2ꢃOH), 7.42 (m, 1H,
Harom), 7.71–7.86 (m, 4H, Harom), 8.14 (dd, 1H, J¼8.4 Hz,
1.1 Hz, Harom), 8.27 (dd, 1H, J¼8.4 Hz, 1.1 Hz, Harom),
8.39 (d, 1H, J¼7.7 Hz, Harom). 13C NMR (DMSO-d6):
d 32.22 (C-20), 38.26 (C-10), 65.81 (C-40), 69.04 (C-30),
110.43, 118.54, 120.88, 122.18, 125.99, 127.51, 128.95,
129.08, 131.37, 138.59, 139.56, 139.93, 144.35, 144.90
(Carom). HRMS (MALDI): m/z calcd for C18H18N3O2+
(MH+): 308.1393, found 308.1395.
4.4.6. 6-(2-Deoxy-b-D-ribofuranosyl)-indolo[2,3-b]qui-
noxaline (8). Sodium (0.056 g, 1 mmol) was dissolved in
methanol (20 mL), and 17 (0.383 g, 0.67 mmol) was added.
The reaction mixture was stirred at rt for three days. The
solution was neutralized with NH4Cl (s), and stirred for
another 30 min. The mixture was filtered and product was
purified by silica gel column chromatography using 5%
MeOH in CH2Cl2 as an eluent.
Yield: 0.165 g (73%) as a yellow solid, Rf 0.35 (8% MeOH/
CH2Cl2), mp 226 ꢁC. IR (KBr): 3368 cmꢂ1 1H NMR
.
(DMSO-d6): d 2.23 (ddd, 1H, J¼13.0 Hz, 6.3 Hz, 2.5 Hz,
H-20), 3.04–3.14 (m, 1H, H-200), 3.72 (dd, 1H, J¼11.6 Hz,
4.5 Hz, H-50), 3.82 (dd, 1H, J¼11.6 Hz, 3.9 Hz, H-500),
3.99 (q, 1H, J¼3.9 Hz, H-40), 4.61–4.64 (m, 1H, H-30),
5.23 (br s, 1H, OH), 5.42 (br s, 1H, OH), 7.01 (dd, 1H,
J¼8.7 Hz, 6.3 Hz, H-10), 7.46 (t, 1H, J¼7.7 Hz, Harom),
7.72–7.88 (m, 3H, Harom), 8.09 (dd, 2H, J¼8.3 Hz, 1.2 Hz,
Harom), 8.28 (dd, 1H, J¼8.3 Hz, 1.2 Hz, Harom), 8.40 (d,
1H, J¼7.7 Hz, Harom). 13C NMR (DMSO-d6): d 36.91
(C-20), 61.76 (C-50), 70.81 (C-30), 82.92 (C-40), 87.10
(C-10), 112.72, 119.28, 121.64, 122.10, 126.63, 127.44,
128.98, 129.20, 131.32, 138.78, 139.32, 139.79, 142.71,
144.61 (Carom). HRMS (MALDI): m/z calcd for
C19H17N3O3Na+ (MNa+): 358.1162, found 358.1151.
4.4.4. (S)-5-(Indolo[2,3-b]quinoxalin-6-yl)-pentane-1,2-
diol (7). Yield: 0.103 g (88%) as yellow crystals (obtained
from 0.132 g, 0.37 mmol of 14d). 1H NMR (CD3OD):
d 1.30–1.39 (m, 1H, H-30), 1.44–1.50 (m, 1H, H-30), 1.79–
1.94 (m, 2H, H-20), 3.20–3.30 (m, 2H, H-50), 3.52 (m, 1H,
H-40), 4.25 (m, 2H, H-10), 7.11–8.14 (m, 8H, Harom). 13C
NMR (CD3OD): d 25.78 (C-20), 31.61 (C-30), 42.32 (C-10),
67.23 (C-50), 72.80 (C-40), 111.03, 119.77, 122.04, 123.43,
127.18, 128.47, 129.44, 129.94, 132.53, 139.48, 140.67,
141.44, 145.71, 146.50 (Carom). HRMS (MALDI): m/z calcd
for C19H19N3O2Na+ (MNa+): 344.1369, found 344.1363.
4.4.5. 6-(20-Deoxy-30,50-di-O-(p-toluoyl)-b-D-ribofurano-
syl)-indolo[2,3-b]quinoxaline (17). NaH (60% in oil,
0.274 g, 6.8 mmol, 1.5 equiv) was added portionwise to a
solution of 6H-indolo[2,3-b]quinoxaline (2, 1.0 g, 4.6 mmol,
1 equiv) in dry DMF (40 mL) under N2. After stirring at rt
4.5. General procedure for DMT-protection of diols 4–8
The diols were dissolved in either dry pyridine (20 mL) (5
and 6) or CH2Cl2 (20 mL) and Et3N (1 mL) (4, 7 and 8),
and DMT–chloride (1.1–1.5 equiv) were added. The

M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
11195
reaction mixture was stirred at rt for 36 h and concentrated
under reduced pressure. The residue was purified by silica
gel column chromatography using EtOAc/cyclohexane/
Et3N (33:65:2 v/v/v) as an eluent.
0.32 mmol of 7), Rf 0.13 (EtOAc/cyclohexane 1:2). 1H
NMR (CDCl3): d 1.51–1.59 (m, 2H, H-30), 1.97–2.13 (m,
2H, H-20), 3.01 (dd, 1H, J¼6.8 Hz, 9.3 Hz, H-50), 3.12 (dd,
1H, J¼3.7 Hz, 9.3 Hz, H-50), 3.74 (s, 6H, 2ꢃOCH3), 3.91–
3.93 (m, 1H, H-40), 4.48–4.57 (m, 2H, H-10), 6.74 (d, 4H,
J¼8.8 Hz, Harom), 7.17 (dd, 2H, J¼2.5 Hz, 6.5 Hz, Harom),
7.23 (d, 6H, J¼8.8 Hz, Harom), 7.35 (d, 2H, J¼7.4 Hz,
Harom), 7.45 (d, 1H, J¼8.1 Hz, Harom), 7.63–7.73 (m, 3H,
Harom), 8.07 (dd, 1H, J¼8.1 Hz, 1.6 Hz, Harom), 8.29 (dd,
1H, J¼8.1 Hz, 1.6 Hz, Harom), 8.47 (d, 1H, J¼7.4 Hz,
41.28 (C-10), 55.12 (2ꢃOCH3), 67.44 (C-50), 70.89 (C-40),
85.90 (CDMT), 109.48, 113.00, 119.41, 120.81, 122.68,
125.88, 126.69, 127.65, 127.71, 128.00, 128.71, 129.23,
129.91, 130.96, 135.86, 135.91, 139.16, 140.03, 140.33,
144.30, 144.72, 145.60, 158.35 (Carom).
4.5.1. (S)-(4,40-Dimethoxytriphenylmethyloxy)-3-indolo-
[2,3-b]quinoxalin-6-yl-propan-2-ol (15a). Yield: 0.147 g
(44%) as a yellow oil (obtained from 0.164 g, 0.56 mmol
1
of 4). H NMR (CDCl3): d 2.81 (br s, 1H, OH), 3.16 (dd,
1H, J¼6.7 Hz, 9.5 Hz, H-30), 3.37 (dd, 1H, J¼5.1 Hz,
9.5 Hz, H-30), 3.73, 3.78 (2ꢃs, 6H, 2ꢃOCH3), 4.39 (m,
1H, H-20), 4.57 (dd, 1H, J¼6.0 Hz, 14.6 Hz, H-10), 4.64
(dd, 1H, J¼3.7 Hz, 14.6 Hz, H-10), 6.72 (d, 1H, J¼8.7 Hz,
Harom), 6.81 (d, 4H, J¼9.1 Hz, Harom), 7.14–7.40 (m, 9H,
Harom), 7.48–7.72 (m, 4H, Harom), 8.01 (dd, 1H, J¼8.2 Hz,
1.3 Hz, Harom), 8.25 (dd, 1H, J¼8.2 Hz, 1.3 Hz, Harom),
8.41 (d, 1H, J¼7.6 Hz, Harom). 13C NMR (CDCl3): d 46.19
(C-10), 55.14 (2ꢃOCH3), 64.91 (C-30), 70.32 (C-20), 86.42
(CDMT), 110.05, 113.03, 113.12, 119.38, 121.12, 122.50,
126.12, 126.77, 127.02, 127.43, 127.78, 127.99, 129.10,
129.24, 129.93, 130.96, 135.68, 135.81, 139.20, 139.62,
140.15, 144.71, 144.82, 145.94, 158.42, 158.59 (Carom).
HRMS (MALDI): m/z calcd for C38H33N3O4Na+ (MNa+):
618.2363, found 618.2379.
13
Harom). C NMR (CDCl3): d 24.95 (C-20), 30.30 (C-30),
4.5.5. 6-[2-Deoxy-5-O-(4,40-dimethoxytriphenylmethyl)-
b-D-ribofuranosyl]-indolo[2,3-b]quinoxaline (18). Yield:
0.148 g (58%) as a yellow oil (obtained from 0.134 g,
1
0.40 mmol of 8), Rf 0.35 (4% MeOH/CH2Cl2). H NMR
(CDCl3): d 2.36–2.46 (m, 2H, H-20), 3.43–3.56 (m, 2H,
H-50), 3.72 (s, 6H, 2ꢃOCH3), 4.16 (m, 1H, H-40), 5.02 (m,
1H, H-30), 6.71 (m, 4H, Harom), 7.00 (t, 1H, J¼7.1 Hz,
H-10), 7.15–7.43 (m, 11H, Harom), 7.66–7.71 (m, 2H, Harom),
7.80 (d, 1H, J¼7.6 Hz, Harom), 7.92 (dd, 1H, J¼7.6 Hz,
2.2 Hz, Harom), 8.26 (dd, 1H, J¼7.6 Hz, 2.2 Hz, Harom),
8.44 (dd, 1H, J¼7.6 Hz, 2.2 Hz, Harom). 13C NMR
(CDCl3): d 37.21 (C-20), 55.15 (2ꢃOCH3), 63.64 (C-50),
72.69 (C-30), 83.09 (C-40), 84.98 (C-10), 86.46 (CDMT),
111.98, 113.05, 120.11, 121.61, 122.57, 126.44, 126.80,
127.77, 127.98, 128.18, 128.72, 129.13, 130.04, 130.09,
130.97, 135.72, 135.79, 139.38, 140.02, 140.33, 143.12,
144.65, 145.19, 158.42, 158.45 (Carom). HRMS (MALDI):
m/z calcd for C40H35N3O5Na+ (MNa+): 660.2469, found
660.2450.
4.5.2. (S)-1-(4,40-Dimethoxytriphenylmethyloxy)-3-(2,3-
dichloro-indolo[2,3-b]quinoxalin-6-yl)-propan-2-ol
(15b). Yield: 0.200 g (62%) as yellow foam (obtained from
1
0.175 g, 0.48 mmol of 5). H NMR (CDCl3): d 3.01 (br s,
1H, OH), 3.24 (dd, 1H, J¼6.1 Hz, 9.7 Hz, H-30), 3.37 (dd,
1H, J¼5.1 Hz, 9.7 Hz, H-30), 3.76, 3.79 (2ꢃs, 6H,
2ꢃOCH3), 4.38–4.41 (m, 1H, H-20), 4.53–4.56 (m, 2H,
H-10), 6.74 (d, 4H, J¼8.7 Hz, Harom), 6.82 (d, 1H, J¼8.7 Hz,
Harom), 7.16–7.69 (m, 11H, Harom), 8.06 (s, 1H, Harom),
8.28 (s, 1H, Harom), 8.59 (d, 1H, J¼4.1 Hz, Harom). 13C
NMR (CDCl3): d 45.87 (C-10), 55.04, 55.15 (2ꢃOCH3),
65.12 (C-30), 70.01 (C-20), 86.47 (CDMT), 110.31, 113.06,
113.11, 118.86, 121.51, 122.75, 123.69, 126.84, 127.01,
127.81, 127.99, 129.11, 129.54, 129.93, 131.65, 132.86,
135.64, 135.93, 137.68, 138.56, 140.85, 144.60, 145.10,
149.76, 158.47 (Carom). HRMS (MALDI): m/z calcd for
C38H32Cl2N3O+4 (MH+): 664.1764, found 664.1791.
4.6. General procedure for synthesis of phosphor-
amidites
The DMT-protected compound (15a–d, 18) was mixed with
diisopropylammoniumtetrazolide (1.7 equiv) and dissolved
in dry CH2Cl2 (10 mL). 2-Cyanoethyl-N,N,N0,N0-tetra-
isopropylphosphane (2.5 equiv) was added, and the reaction
mixture was stirred at rt overnight. The solvent was
evaporated off under reduced pressure, and the residue was
purified by silica gel column chromatography using
EtOAc/cyclohexane/Et3N (49:49:2 v/v/v) as an eluent.
4.5.3. (S)-1-(4,40-Dimethoxytriphenylmethyloxy)-4-
indolo[2,3-b]quinoxalin-6-yl-butan-2-ol (15c). Yield:
0.206 g (83%) as a yellow oil (obtained from 0.125 g,
1
0.41 mmol of 6). H NMR (CDCl3): d 1.71–2.22 (m, 2H,
H-20), 3.04–3.19 (m, 3H, H-30, H-40), 3.55 (br s, 1H, OH),
3.73, 3.79 (2ꢃs, 6H, 2ꢃOCH3), 4.46 (m, 1H, H-10), 4.80
(m, 1H, H-10), 6.71–6.86 (m, 4H, Harom), 7.11–7.53 (m,
11H, Harom), 7.66–7.78 (m, 3H, Harom), 8.09 (dd, 1H,
J¼8.1 Hz, 1.5 Hz, Harom), 8.31 (dd, 1H, J¼8.1 Hz, 1.5 Hz,
Harom), 8.49 (d, 1H, J¼7.3 Hz, Harom). 13C NMR (CDCl3):
d 32.92 (C-20), 37.97 (C-10), 55.13, 55.22 (2ꢃOCH3),
67.29 (C-30, C-40), 85.98 (CDMT), 109.45, 113.00, 119.51,
121.12, 122.79, 126.11, 126.66, 127.03, 127.50, 127.68,
127.74, 127.81, 128.09, 128.97, 129.11, 129.35, 129.92,
131.18, 135.99, 139.30, 139.45, 139.87, 144.37, 144.76,
158.33 (Carom).
4.6.1. Phosphoramidite of (S)-1-(4,40-dimethoxytriphe-
nylmethyloxy)-3-indolo[2,3-b]quinoxalin-6-yl-propan-2-
ol (16a). Yield: 0.078 g (61%) as a yellow oil (obtained from
0.095 g, 0.16 mmol of 15a), Rf 0.58 (EtOAc/cyclohexane
1:1). 31P NMR (CDCl3): d 149.96, 150.27. HRMS
(MALDI): m/z calcd for C47H50N5O5PNa+ (MNa+):
818.3442, found 818.3401.
4.6.2. Phosphoramidite of (S)-1-(4,40-dimethoxytriphe-
nylmethyloxy)-3-(2,3-dichloro-indolo[2,3-b]quinoxalin-
6-yl)-propan-2-ol (16b). Yield: 0.146 g (66%) as a yellow
oil (obtained from 0.170 g, 0.26 mmol of 15b), Rf 0.64
4.5.4. (S)-1-(4,40-Dimethoxytriphenylmethyloxy)-5-
indolo[2,3-b]quinoxalin-6-yl-pentan-2-ol (15d). Yield:
0.068 g (34%) as a yellow oil (obtained from 0.103 g,
1
(EtOAc/cyclohexane 1:1). H NMR (CDCl3): d 0.74–1.28
(m, 12H, 4ꢃCH3), 2.06, 2.32 (2ꢃt, 2H, J¼6.6 Hz, CH2CN),

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M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
3.22–3.60 (m, 6H, H-30, OCH2CH2CN, 2ꢃCH(CH3)2), 3.77
(s, 6H, 2ꢃOCH3), 4.07–4.16 (m, 1H, H-20), 4.62–4.67 (m,
2H, H-10), 6.76–6.84 (m, 4H, Harom), 7.16–7.69 (m, 12H,
Harom), 8.12–8.41 (m, 3H, Harom). 13C NMR (CDCl3):
13C NMR (CDCl3): d 20.33 (CH2CN), 24.52, 24.54, 24.60,
24.67 (4ꢃCH(CH3)2), 36.39 (C-20), 43.21, 43.32
(2ꢃCH(CH3)2), 55.15 (2ꢃOCH3), 58.53 (OCH2CH2CN),
62.98 (C-50), 73.32 (C-30), 83.29 (C-40), 84.73 (C-10),
86.30 (CDMT), 112.29, 112.97 (Carom), 117.45 (CN),
120.14, 121.58, 122.47, 126.37, 126.76, 127.68, 127.93,
128.31, 128.68, 129.21, 130.17, 130.97, 135.70, 135.74,
135.78, 135.81, 139.45, 140.04, 143.11, 144.64, 145.22,
158.39 (Carom). 31P NMR (CDCl3): d 149.63, 150.02.
HRMS (MALDI): m/z calcd for C49H52N5O6PNa+
(MNa+): 860.3547, found 860.3578.
d
20.09 (CH2CN), 24.28, 24.40, 24.46, 24.54
(4ꢃCH(CH3)2), 42.73, 42.92 (2ꢃCH(CH3)2), 44.42 (C-10),
55.17 (2ꢃOCH3), 57.75 (OCH2CH2CN), 64.87 (C-30),
70.63 (C-20), 86.28 (CDMT), 110.57, 113.02 (Carom),
119.08 (CN), 121.29, 122.76, 126.76, 127.73, 128.11,
128.27, 129.10, 129.70, 129.98, 131.50, 132.60, 135.79,
135.98, 137.83, 139.27, 144.71, 145.10, 145.93, 158.43
(Carom). 31P NMR (CDCl3): d 150.20, 150.34. HRMS
(ESI): m/z calcd for C47H48N5O5Cl2PNa+ (MNa+):
886.2662, found 886.2684.
4.6.6. 6-Methylindolo[2,3-b]quinoxaline-3-carboxylic
acid methyl ester (21). 6H-Indolo[2,3-b]quinoxaline-3-
carboxylic acid58 (20, 2.50 g, 9.5 mmol) was suspended in
dry DMSO (25 mL) and powdered KOH (1.85 g, 33 mmol,
3.5 equiv) was added. The reaction mixture turned red, as
it was stirred for 15 min CH3I (5.11 g, 36 mmol, 3.8 equiv)
was added through a syringe. The reaction mixture was
stirred at rt for 48 h. N2 was bubbled through the reaction
mixture in order to get rid of excess of CH3I. H2O
(50 mL) was added, and the compound precipitated. The
yellow powder was filtered, washed with water and dried
in vacuo. A small sample was separated for analysis by silica
gel column chromatography using EtOAc/PE 1:1 as an
eluent. The rest was used for the next step without further
purification. The ratio between the ester and the carboxylic
acid was 3:2.
4.6.3. Phosphoramidite of (S)-1-(4,40-dimethoxytriphe-
nylmethyloxy)-4-indolo[2,3-b]quinoxalin-6-yl-butan-2-ol
(16c). Yield: 0.206 g (84%) as a yellow oil (obtained from
1
0.185 g, 0.30 mmol of 15c). H NMR (CDCl3): d 1.04–
1.26 (m, 12H, 4ꢃCH3), 2.23–2.38 (m, 2H, H-20), 2.40,
2.59 (2ꢃt, 2H, J¼6.5 Hz, CH2CN), 3.10, 3.24 (2ꢃdd, 1H,
J¼6.2 Hz, 9.2 Hz, H-40), 3.36 (m, 1H, H-40), 3.55–3.71
(m, 4H, OCH2CH2CN, 2ꢃCH(CH3)2), 3.77, 3.79 (2ꢃs,
6H, 2ꢃOCH3), 4.51–4.61 (m, 2H, H-10), 4.17–4.22 (m,
1H, H-30), 6.76–6.84 (m, 4H, Harom), 7.16–7.76 (m, 14H,
Harom), 8.04 (m, 1H, Harom), 8.29 (dd, 1H, J¼8.1 Hz,
1.3 Hz, Harom), 8.47 (d, 1H, J¼7.6 Hz, Harom). 31P NMR
(CDCl3): d 149.47, 149.70.
4.6.4. Phosphoramidite of (S)-1-(4,40-dimethoxytriphe-
nylmethyloxy)-5-indolo[2,3-b]quinoxalin-6-yl-pentan-2-
ol (16d). Yield: 0.066 g (73%) as a yellow oil (obtained from
0.068 g, 0.11 mmol of 15d), Rf 0.58 (EtOAc/cyclohexane
Yellow solid, Rf 0.53 (EtOAc/PE 1:1), mp 180 ꢁC. 1H NMR
(CDCl3): d 3.87, 4.00 (2ꢃs, 6H, 2ꢃCH3), 7.35 (d, 2H,
J¼7.3 Hz, Harom), 7.66 (t, 1H, J¼7.3 Hz, Harom), 8.21 (m,
2H, Harom), 8.37 (d, 1H, J¼7.2 Hz, Harom), 8.73 (s, 1H,
Harom). 13C NMR (CDCl3): d 27.39 (CH3), 52.34 (OCH3),
109.20, 118.88, 121.10, 122.91, 125.31, 129.30, 129.61,
130.30, 131.65, 139.45, 141.12, 141.38, 145.32, 145.88
(Carom), 166.71 (COOR). HRMS (MALDI): m/z calcd for
C17H14N3O+2 (MH+): 292.1081, found 292.1071.
1
1:1). H NMR (CDCl3): d 1.69–2.00 (m, 2H, H-30), 2.34,
2.64 (2ꢃt, 2H, J¼6.5 Hz, CH2CN), 2.90 (dd, 1H, J¼
6.2 Hz, 9.2 Hz, H-50), 3.01–3.15 (m, 1H, H-50), 3.41–3.51,
3.56–3.70 (2ꢃm, 4H, OCH2CH2CN, 2ꢃCH(CH3)2), 3.73
(s, 6H, 2ꢃOCH3), 3.97–4.06 (m, 1H, H-40), 4.47–4.53 (m,
2H, H-10), 6.71 (m, 4H, Harom), 7.13–7.75 (m, 14H, Harom),
8.07 (m, 1H, Harom), 8.29 (dd, 1H, J¼8.1 Hz, 1.2 Hz, Harom),
8.48 (d, 1H, J¼7.6 Hz, Harom). 13C NMR (CDCl3): d 20.29
(CH2CN), 23.71 (C-20), 24.36, 24.46, 24.55, 24.66
(4ꢃCH3), 30.85 (C-30), 41.35 (C-10), 42.87, 43.04
(2ꢃCH(CH3)2), 55.12 (2ꢃOCH3), 58.09 (OCH2CH2CN),
65.66 (C-50), 72.70 (C-40), 85.76 (CDMT), 109.52, 112.91,
117.61, 119.44, 120.75, 122.66, 125.85, 126.55, 126.62,
127.62, 127.77, 127.81, 128.04, 128.11, 128.65, 129.31,
129.95, 130.91, 136.00, 139.21, 140.01, 144.34, 144.46,
144.86, 158.26, 158.31 (Carom). 31P NMR (CDCl3):
d 149.08, 149.60. HRMS (MALDI): m/z calcd for
C49H54N5O5PNa+ (MNa+): 846.3755, found 846.3778.
4.6.7. 6-Methylindolo[2,3-b]quinoxaline-3-carboxylic
acid (22). Yellow solid, Rf 0.42 (EtOAc/PE 1:1), mp
192 ꢁC. 1H NMR (DMSO-d6): d 3.78 (s, 3H, CH3), 7.34 (t,
1H, J¼7.5 Hz, Harom), 7.57 (d, 1H, J¼8.0 Hz, Harom), 7.71
(t, 1H, J¼7.5 Hz, Harom), 7.93–8.14 (m, 2H, Harom), 8.22
(d, 1H, J¼7.5 Hz, Harom), 8.46 (s, 1H, Harom). 13C NMR
(DMSO-d6): d 27.30 (CH3), 110.06, 117.98, 120.96,
122.26, 124.95, 129.04, 129.28, 130.29, 131.79, 138.78,
140.21, 140.64, 145.04, 145.17 (Carom), 166.96 (COOH).
HRMS (MALDI): m/z calcd for C16H11N3O+2 (MH+):
278.0924, found 278.0922.
4.6.8. (6-Methylindolo[2,3-b]quinoxalin-3-yl)methanol
(23). LiAlH4 (0.65 g, 17.1 mmol) was suspended in dry
THF (20 mL) at 0 ꢁC under N2. The crude mixture of 21
and 22 was dissolved in dry THF (20 mL) and added
dropwise using a syringe. The icebath was removed and
the reaction was gently refluxed for 5 h. The reaction was
quenched at 0 ꢁC by carefully adding 1 mL H2O, 1 mL of
a 15% aq solution of NaOH and 3 mL of H2O. The reaction
mixture was filtered and washed with ethanol. The solvent
was evaporated off under reduced pressure, and the residue
was purified by silica gel column chromatography using
5% MeOH/CHCl3 as an eluent.
4.6.5. Phosphoramidite of 6-[2-deoxy-5-O-(4,40-dimeth-
oxytriphenylmethyl)-b-D-ribofuranosyl]-indolo[2,3-
b]quinoxaline (19). Yield: 0.033 g (21%) as a yellow oil
(obtained from 0.119 g, 0.19 mmol of 18). 1H NMR
(CDCl3): d 1.19–1.28 (m, 12H, 4ꢃCH3), 2.47, 2.63 (2ꢃt,
2H, J¼6.5 Hz, CH2CN), 3.41–3.92 (m, 8H, H-20, H-50,
OCH2CH2CN, 2ꢃCH(CH3)2), 3.73 (2ꢃs, 6H, 2ꢃOCH3),
4.30 (m, 1H, H-40), 5.04–5.18 (m, 1H, H-30), 6.66–6.72
(m, 4H, Harom), 7.01–7.43 (m, 12H, H-10, Harom), 7.70
(m, 2H, Harom), 7.89–7.96 (m, 2H, Harom), 8.27 (dd,
1H, J¼7.6 Hz, 2.2 Hz, Harom), 8.43–8.46 (m, 1H, Harom).

M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
11197
Yield: 1.10 g (44% over two steps) as yellow crystals,
Rf 0.34 (5% MeOH/CHCl3), mp 175 ꢁC. IR (KBr):
125.76, 129.40, 130.90, 137.90, 138.67, 139.35, 140.39,
144.84, 145.68 (Carom). HRMS (MALDI): m/z calcd for
C23H25N3O3Na+ (MNa+): 414.1788, found 414.1776.
1
3371 cmꢂ1. H NMR (DMSO-d6): d 3.89 (s, 1H, CH3),
4.80 (d, 2H, J¼5.4 Hz, CH2), 5.53 (t, 1H, OH), 7.39 (t,
1H, J¼7.7 Hz, Harom), 7.66–7.80 (m, 3H, Harom), 8.03 (s,
1H, Harom), 8.20 (d, 1H, J¼8.7 Hz, Harom), 8.35 (d, 1H,
J¼7.7 Hz, Harom). 13C NMR (DMSO-d6): d 27.36 (CH3),
62.77 (CH2), 110.01, 118.44, 120.78, 121.81, 123.78,
124.91, 128.61, 130.96, 137.59, 138.80, 139.83, 143.71,
144.44, 145.16 (Carom). HRMS (MALDI): m/z calcd for
C16H14N3O+ (MH+): 264.1131, found 264.1128.
4.6.11. (S)-4-(6-Methylindolo[2,3-b]quinoxalin-3-yl-
methoxy)-butane-1,2-diol (9). The same procedure was
used as for compounds 4–7.
Quantitative yield. Yellow oil. 1H NMR (DMSO-d6): d 1.53–
1.62, 1.80–1.90 (2ꢃm, 2H, H-20), 3.23–3.32 (m, 2H, H-40),
3.66 (t, 2H, J¼6.5 Hz, H-10), 3.82–3.95 (m, 1H, H-30),
3.89 (s, 3H, NCH3), 4.51 (br s, 2H, 2ꢃOH), 4.73 (s, 2H,
CH2), 7.39 (t, 1H, J¼7.3 Hz, Harom), 7.62–7.83 (m, 3H,
Harom), 7.99–8.13 (m, 1H, Harom), 8.19 (d, 1H, J¼8.6 Hz,
Harom), 8.33 (d, 1H, J¼7.6 Hz, Harom). 13C NMR (DMSO-
d6): d 27.40 (CH3), 33.67 (C-20), 66.04 (C-10), 67.13
(C-40), 68.44 (C-30), 71.47 (CH2), 110.09, 118.40, 120.83,
121.89, 124.94, 125.31, 128.83, 131.09, 137.77, 139.10,
139.68, 139.90, 144.58, 145.21 (Carom). HRMS (MALDI):
m/z calcd for C20H21N3O3Na+ (MNa+): 374.1475, found
374.1462.
4.6.9. 3-Chloromethyl-6-methylindolo[2,3-b]quinoxaline
(24). (6-Methylindolo[2,3-b]quinoxalin-3-yl)methanol (23,
0.500 g, 1.9 mmol) was suspended in a mixture of pyridine
(0.23 mL) and CH2Cl2 (10 mL) and the suspension was
cooled to 0 ꢁC. SOCl2 (0.25 mL) was added slowly with a
syringe. The reaction was stirred at rt overnight. The reaction
mixture was poured into water (20 mL) and CH2Cl2 (10 mL)
was added. The two-phase system was stirred for 30 min,
after which the phases were separated. The organic phase
was washed with 5% NaHCO3 (aq) (2ꢃ25 mL) and satd
aq NaCl (2ꢃ25 mL) and dried using MgSO4. The solvent
was removed under reduced pressure yielding a yellow solid,
which was purified by silica gel column chromatography
using 1% MeOH/CH2Cl2 as eluent.
4.6.12. (S)-1-(4,40-Dimethoxytriphenylmethyloxy)-4-(6-
methylindolo[2,3-b]quinoxalin-3-ylmethoxy)-butan-2-ol
(26). The same procedure was used as for DMT-protection of
compounds 4–8.
1
Yield: 0.478 g (89%) as a yellow solid, Rf 0.50 (1% MeOH/
CH2Cl2), mp 204–205 ꢁC. 1H NMR (CD2Cl2): d 3.84 (s, 3H,
CH3), 4.78 (s, 2H, CH2), 7.30 (t, 1H, J¼7.4 Hz, Harom), 7.40
(d, 1H, J¼8.2 Hz, Harom), 7.59–7.64 (m, 2H, Harom), 8.00 (s,
1H, Harom), 8.13 (d, 1H, J¼8.6 Hz, Harom), 8.32 (d, 1H,
J¼7.9 Hz, Harom). 13C NMR (CD2Cl2): d 27.76 (CH3),
46.57 (CH2), 109.83, 119.34, 121.52, 122.92, 126.71,
127.44, 129.66, 129.87, 131.80, 138.66, 138.84, 140.54,
140.79, 145.60 (Carom). HRMS (MALDI): m/z calcd for
C16H13ClN+3 (MH+): 282.0973, found 282.0795.
Yield: 0.149 g (55%) as a yellow oil, H NMR (CDCl3):
d 1.80–1.86 (m, 2H, H-20), 3.13–3.17, 3.68–3.73 (2ꢃm,
4H, H-10, H-40), 3.76 (s, 6H, 2ꢃOCH3), 3.96 (s, 3H,
NCH3), 4.02–4.12 (m, 1H, H-30), 4.74 (s, 2H, CH2), 6.81
(d, 4H, J¼8.7 Hz, Harom), 7.19–7.29 (m, 3H, Harom), 7.32
(d, 4H, J¼8.7 Hz, Harom), 7.36–7.46 (m, 4H, Harom), 7.59
(dd, 1H, J¼8.5 Hz, 1.8 Hz, Harom), 7.70 (t, 1H, J¼7.0 Hz,
Harom), 7.98–8.20 (m, 1H, Harom), 8.25 (d, 1H, J¼8.7 Hz,
Harom), 8.46 (d, 1H, J¼7.8 Hz, Harom). 13C NMR (CDCl3):
d 27.50 (CH3), 33.53 (C-20), 55.16 (OCH3), 67.33 (C-10),
68.14 (C-40), 69.43 (C-30), 72.95 (CH2), 85.97 (CDMT),
109.16, 113.06, 120.88, 120.95, 122.58, 125.47, 125.85,
126.72, 127.77, 128.14, 129.42, 130.02, 130.93, 136.05,
138.67, 139.21, 140.38, 144.85, 158.41 (Carom). HRMS
(MALDI): m/z calcd for C20H39N3O5Na+ (MNa+):
676.2782, found 676.2757.
4.6.10. 3-[2-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)ethoxy-
methyl]-6-methylindolo[2,3-b]quinoxaline (25). To a solu-
tion of 2-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-ethanol
(0.180 g, 1.23 mmol) in dry toluene (15 mL) was added
pulverized KOH (0.736 g, 13.0 mmol) followed by 3-chloro-
methyl-6-methylindolo[2,3-b]quinoxaline (24, 0.205 g,
0.73 mmol). A Dean–Stark apparatus was filled with dry tol-
uene in the side arm and used, while refluxing the reaction
mixture for 48 h. H2O (10 mL) was added and the organic
phase was washed with H2O (3ꢃ10 mL), dried using
MgSO4 and evaporated under reduced pressure yielding
a yellow oil, which was purified by silica gel column chro-
matography using 2% MeOH/CH2Cl2 as an eluent.
4.6.13. Phosphoramidite of (S)-1-(4,40-dimethoxytri-
phenylmethyloxy)-4-(6-methylindolo[2,3-b]quinoxalin-
3-ylmethoxy)-butan-2-ol (27). The same procedure was
used as for phosphitylation of compounds 15a–d.
Yield: 0.061 g (47%) as a yellow oil, Rf 0.52 (EtOAc/cyclo-
hexane 1:1). 1H NMR (CDCl3): d 1.04–1.17 (m, 12H,
4ꢃCH3), 2.38, 2.53 (2ꢃt, 2H, J¼6.5 Hz, CH2CN), 1.92–
1.99 (m, 1H, H-20), 2.08–2.17 (m, 1H, H-20), 3.02–3.07,
3.20–3.23 (2ꢃm, 2H, H-40), 3.49–3.72 (m, 6H, H-10,
OCH2CH2CN, 2ꢃCH(CH3)2), 3.76 (s, 6H, 2ꢃOCH3), 3.98
(s, 3H, NCH3), 4.19–4.22 (m, 1H, H-30), 4.70–4.74 (m,
2H, CH2), 6.77–6.82 (m, 4H, Harom), 7.16–7.29 (m, 4H,
Harom), 7.34 (d, 4H, J¼8.8 Hz, Harom), 7.40 (t, 1H, J¼
7.5 Hz, Harom), 7.46 (dd, 2H, J¼2.9 Hz, 7.3 Hz, Harom),
7.62 (m, 1H, Harom), 7.71 (t, 1H, J¼7.3 Hz, Harom), 8.06
(d, 1H, J¼8.6 Hz, Harom), 8.25 (dd, 1H, J¼1.0 Hz, 8.6 Hz,
Harom), 8.47 (d, 1H, J¼7.5 Hz, Harom). 13C NMR (CDCl3):
d 21.03 (CH2CN), 24.43, 24.54, 24.60, 24.73 (4ꢃCH3),
Yield: 0.190 g (67%) as a yellow oil, Rf 0.46 (4% MeOH/
CH2Cl2). 1H NMR (CDCl3): d 1.37, 1.41 (2ꢃs, 6H,
2ꢃCH3), 1.91–1.97 (m, 2H, H-20), 3.59–3.64 (m, 1H,
H-30), 3.69 (t, 2H, J¼6.8 Hz, H-10), 3.94 (s, 3H, NCH3),
4.08–4.13 (m, 1H, H-40), 4.26–4.30 (m, 1H, H-400), 4.76 (s,
2H, CH2), 7.34–7.43 (m, 2H, Harom), 7.62–7.70 (m, 2H,
Harom), 7.97–8.21 (m, 1H, Harom), 8.25 (d, 1H, J¼8.6 Hz,
Harom), 8.44 (d, 1H, J¼7.7 Hz, Harom). 13C NMR (CDCl3):
d 25.78, 26.95 (C(CH3)2), 27.48 (CH3), 33.92 (C-20), 67.35
(C-10), 69.67 (C-40), 72.85 (CH2), 73.80 (C-30), 108.58
(C(CH3)2), 109.14, 119.35, 120.93, 122.54, 125.43,

11198
M. C. Wamberg et al. / Tetrahedron 62 (2006) 11187–11199
26.90 (CH3), 33.84 (C-20), 42.33, 42.81 (2ꢃCH(CH3)2),
55.15 (2ꢃOCH3), 58.23 (OCH2CH2CN), 64.61 (C-10),
66.31 (C-40), 67.08 (C-30), 72.72 (CH2), 85.86 (CDMT),
109.19, 112.97 (Carom), 119.33 (CN), 120.95, 122.57,
125.56, 125.77, 126.59, 127.67, 128.24, 129.34, 130.16,
130.92, 136.19, 138.03, 139.27, 140.44, 144.06, 144.99,
158.41 (Carom). 31P NMR (CDCl3): d 148.57, 149.06.
HRMS (MALDI): m/z calcd for C50H56N5O6PNa+
(MNa+): 876.3860, found 876.3865.
1 mM EDTA buffer at pH¼7.0 with a concentration of
1.5 mM of each strand.
4.9. Single-crystal X-ray diffraction
Crystals of 4 and 23$H2O were obtained from solutions in
MeOH/CH2Cl2, layered with hexane. X-ray diffraction
data for 4 were collected at Beamline I911-3 at the MAX-II
storage ring, MAX-lab, University of Lund, Sweden
˚
(l¼0.750 A). Diffraction data for 23$H2O were collected
Crystallographic data (excluding structure factors) for the
structures in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC-609476 (4) and CCDC-
609477 (23$H2O). Copies of the data can be obtained, free
of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK, (fax: +44 1223 36033 or
e-mail: deposit@ccdc.cam.ac.uk).
locally with a Bruker Nonius X8APEX-II instrument.
Molecule 4 crystallizes in the non-centrosymmetric space
group P21 with two molecules in the asymmetric unit. The
ring systems adopt a centrosymmetric arrangement (space
group P21/n), but the centrosymmetry is broken by the chiral
side-chains. It was not possible to determine the absolute
structure: the S enantiomer was assigned on the basis of
the chemistry and Friedel opposites were merged as equiva-
lent data in the final cycles of refinement.
4.7. ODN and INA syntheses, purification and
measurement of melting temperatures
Acknowledgements
The ODN and INA syntheses were carried out on an
ExpediteÔ Nucleic Acid Synthesis System Model 8909
from Applied Biosystems. The 6H-indolo[2,3-b]quinoxaline
amidits 16a–d, 19 and 27 were dissolved in dry MeCN (if
necessary a 1:1 mixture of dry MeCN and dry CH2Cl2), as
a 0.075 M solution and inserted into the growing oligo-
nucleotide chain using the same conditions as for normal
nucleotide couplings (2 min coupling). The ODNs were syn-
thesized with DMT and purified on a Waters Prep LC 4000
HPLC with a Waters Prep LC controller and a Waters
2487 Dual l Absorbance detector on a Waters XterraÔ
MS C18 column. Buffer A [950 mL of 0.1 M NH4HCO3
and 50 mL of MeCN (pH¼9.0)] and buffer B [250 mL of
0.1 M NH4HCO3 and 750 mL of MeCN (pH¼9.0)]. Gradi-
ents: 5 min 100% A, linear gradient to 70% B in 30 min,
2 min with 70% B, linear gradient to 100% B in 8 min and
then 100% A in 15 min (product peak at w35 min). The
ODNs were DMT-deprotected in 80% aq acetic acid
(100 mL) for 20 min, diluted with 1 M sodium acetate
(150 mL), and precipitated from abs ethanol (600 mL). All
modified ODNs were confirmed by MALDI-TOF analysis
on a Voyager Elite Bio Spectrometry Research Station
from Perceptive Biosystems.
We are grateful to Ulf B. Christensen for introducing us to
oligonucleotide synthesis and purification. Furthermore we
are grateful to the Danish Natural Science Research Council
(SNF) and Carlsbergfondet (Denmark) for granting the
X-ray equipment. Synchrotron access was provided by
MAX-lab, University of Lund, Sweden. Krister Larsson
and Christer Svensson provided valuable assistance with
X-ray data collection and processing.
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