Sulfonamide bearing oligonucleotides: Simple synthesis and efficient RNA recognition

Article abstract of DOI:10.1016/j.bmc.2012.04.036

Four pyrimidine nucleosides wherein a benzensulfonamide group is linked to the C-5 position of the uracil nucleobase through a triazolyl or an alkynyl linker were prepared by Cu(I)-assisted azide-alkyne cycloadditions (CuAAC) or Sonogashira reactions, res

Full text of DOI:10.1016/j.bmc.2012.04.036

Bioorganic & Medicinal Chemistry 20 (2012) 3843–3849
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Sulfonamide bearing oligonucleotides: Simple synthesis and efficient
RNA recognition
Pawan Kumar a,b, Navneet Chandak a, Poul Nielsen b, Pawan K. Sharma a,
⇑
a Department of Chemistry, Kurukshetra University, Kurukshetra 136 119, India
b Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 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:
Four pyrimidine nucleosides wherein a benzensulfonamide group is linked to the C-5 position of the ura-
cil nucleobase through a triazolyl or an alkynyl linker were prepared by Cu(I)-assisted azide-alkyne cyc-
loadditions (CuAAC) or Sonogashira reactions, respectively, and incorporated into oligonucleotides.
Received 12 March 2012
Revised 14 April 2012
Accepted 16 April 2012
Available online 23 April 2012
Efficient p–p-stacking between two or more phenyltriazoles in the major groove was found to increase
the thermal stability of a DNA:RNA duplex significantly. On the other hand, the alkynyl group was not as
efficient in stacking as the triazolyl group. No effect of positional orientation of the sulfonamide group on
the stacking efficiency was observed, and the most stable DNA:RNA duplex contained four consecutive
sulfonamide substituted phenyltriazole moieties in the major groove.
Keywords:
Oligonucleotides
p–p-Stacking
CuAAC reaction
Click chemistry
Sonogashira coupling
RNA-targeting
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
exhibited a decrease in thermal melting by 2.5 °C. Molecular
modelling studies indicated that the duplex with the sulfonamide
The era of nucleic acid therapies was born in 1978 with the
proof of concept for the antisense mechanism whereby a string
of DNA nucleotides could be used to bind and block mRNA func-
tion.1 Thus, antisense technology presents an opportunity to dis-
rupt the gene expression at the translational level by an accurate
knowledge of the target mRNA sequence and rational design of
its complementary antisense oligonucleotide sequence having
higher affinity for RNA so as to form a stable DNA:RNA duplex.2
monomer demonstrated a perfect stacking of the aromatic ring
and a consistent organisation between the neighbouring sulfon-
amide groups.4 In another study, we found that phenyltriazoles
positioned on the 5-positions of cytosines also can participate in
stacking.5 Importantly, the phenyltriazole modified oligonucleo-
tides were found to be very resistant towards nucleolytic
degradation.5 In the present study, we explore the scope of the
stacking effect concerning the positional orientation of a sulfon-
amide group as well as comparing the stacking potential of the
triazole versus an alkynyl linker.
The idea of taking advantage of efficient p–p-stacking for increas-
ing the duplex stability has been exploited by us3–5 and others.6–8
We reported that a triazole group attached to the 5-position of dU
projects itself in the major groove of DNA:DNA or DNA:RNA
duplexes and can lead to increased thermal stabilities through effi-
2. Results and discussion
2.1. Chemical synthesis
cient p–p
-stacking.3 Furthermore, only two consecutive incorpora-
tions were found to be sufficient to give significant DNA:RNA
duplex stabilization due to stacking of triazoles and the aromatic
substituents attached.4 The most pronounced effect was given by
the monomer containing a phenyltriazole with a distal sulfon-
amide moiety.4 Hence a 9-mer DNA:RNA duplex with two p-sul-
fonamidophenyltriazoles (W in Scheme 1) in the center displayed
an increase in melting temperature of 8 °C as compared to the
unmodified duplex while the same 9-mer DNA:DNA duplex
The C5-triazole-functionalized pyrimidine nucleosides were
prepared by following the concept of Click chemistry9 through
Cu(I)-catalyzed azide–alkyne cycloadditions10 performed on 50-O-
DMTr-protected 5-ethynyl-20-deoxyuridine 1 (Scheme 1) using
azides that were prepared and isolated from suitable building
blocks (Scheme 2). In this way, nucleoside 2 was made from the
protected p-azidobenzenesulfonamide 7a as recently published.4
The novel m-azidobenzenesulfonamide 6b was prepared by a dia-
zotation of known metanilamide11 and was protected with a dime-
thylamidine group to give 7b following our successful experience
⇑
Corresponding author. Tel.: +91 94164 57355; fax: +91 1744 238277.
E-mail address: pksharma@kuk.ac.in (P.K. Sharma).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.036

3844
P. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 3843–3849
R'
R
R
R'
O
O
N
NH
N
N
O
N
NH
O
N
N
N
O
NH
O
NH
DMTrO
DMTrO
c
a or b
O
O
O
O
DMTrO
DMTrO
c
a or b
O
OH
O
OH
R = H, R' = SO2N=CHN(CH3)2
9 R = SO2N=CHN(CH3)2, R' = H
8
1
OH
OH
2 R = H, R' = SO2N=CHN(CH3)2
3 R = SO2N=CHN(CH3)2, R' = H
1
R
R
R'
R'
R
R
R'
R'
O
N
O
N
N
N
N
NH
NH
O
N
O
N
N
N
O
O
NH
O
NH
DMTrO
O
d
O
O
N
O
DMTrO
O
d
O
O
O
O
P
O
P
O
(i-Pr)2N
O(CH2)2CN
O
O
P
O
P
O
Y R = H, R' = SO2NH2
R = SO2NH2, R' = H
(i-Pr)2N
O(CH2)2CN
10 R = H, R' = SO2N=CHN(CH3)2
11
Z
R = SO2N=CHN(CH3)2, R' = H
4
W
R = H, R' = SO2N=CHN(CH3)2
R = H, R' = SO2NH2
Scheme 3. Reagents and conditions: (a) Iodophenyl 7c, Pd(PPh3)4, CuI, Et3N, DMF,
65% 8; (b) Iodophenyl 7d, Pd(PPh3)4, CuI, Et3N, DMF, 65% 3; (c) NC(CH2)2OPCl-
N(iPr)2, EtN(iPr)2, CH2Cl2, 67% 10, 73% 11; (d) automated DNA-synthesis.
DMTr = 4,40-dimethoxytrityl.
5 R = SO2N=CHN(CH3)2, R' = H
X R = SO2NH2, R' = H
Scheme 1. Reagents and conditions: (a) Ref.4; (b) Azide 7b, CuSO4, Na ascorbate, t-
BuOH, H2O, THF, 51% 3; (c) NC(CH2)2OPClN(iPr)2, EtN(iPr)2, CH2Cl2, 67% 4,4 80% 5;
(d) automated DNA-synthesis. DMTr = 4,40-dimethoxytrityl.
Table 1
Hybridisation data for DNA:DNA duplexesa
Tm (D
Tm/mod.)b (°C)
R
R
a
B=
W
X
Y
Z
O
O
50-dGTG TBT TGC
50-dGTG TBB TGC
50-dGTG BBB TGC
50-dGTG BBB BGC
28.0c
(–5.0)
30.5c
(–1.3)
33.0c
(0.0)
28.0
(–5.0)
29.5
(–1.8)
32.5
(–0.2)
36.5
31.5
(–1.5)
30.0
(–1.5)
31.0
(–0.7)
31.0
31.5
(–1.5)
30.5
(–1.3)
29.5
(–1.2)
29.5
S
S
ON1
ON2
ON3
ON4
NH2
N
N
O
O
6a R = 4-N3
7a R = 4-N3
6b
7b
R = 3-N3
R = 3-N3
6c R = 4-I
6d R = 3-I
7c R = 4-I
7d R = 3-I
35.5c
(+0.7)
Scheme 2. Reagents and conditions: (a) DMF, POCl3, 73% 7a,4 69% 7b, 65% 7c, 82%
7d.
(+0.9)
(–0.5)
(–0.9)
a
Target sequence 50-dGCA AAA CAC.
Melting temperatures (Tm values/°C) obtained from the maxima of the first
b
derivatives of the melting curves (A260 vs. temperature) recorded in a medium salt
buffer (Na2HPO4 (5 mM), NaCl (100 mM), EDTA (0.1 mM), pH 7.0) using 1.5
concentrations of each strand. In brackets the changes in melting temperature for
each modification Tm/mod./°C) as compared to the unmodified reference
duplex (Tm = 33.0 °C).
l
M
of using this base-sensitive group for p-azidobenzenesulfonamide
6a.4 The protected triazole nucleoside 3 was prepared in good yield
by the Cu(I)-catalyzed cycloaddition of the protected azide 7b with
1 (Scheme 1). Both nucleosides 2 and 3 were converted to the cor-
responding phosphoramidites 44 and 5 in reasonable yields by
using a standard method.
B (D
c
Data taken from Ref. 4
synthesizer with tetrazole as the activator. Standard conditions
were employed except for extended coupling (15 min) for the
modified phosphoramidites. After completion of the synthesis,
the oligonucleotides were removed from the solid support by
treatment with concentrated aqueous ammonia. This treatment
also removed the amidine protections of the sulfonamides giving
the incorporated monomers W, X, Y and Z, respectively (Schemes
1 and 3). The four monomers were incorporated into the same
series of 9-mer oligonucleotides ON1–4 (Tables 1 and 2). The com-
position and purity (>80%) was verified by MALDI-TOF MS analysis
and RP-HPLC, respectively.
The C5-alkynyl-functionalized building blocks 8 and 9 were
prepared in good yields through Sonogashira couplings of 1
(Scheme 3) with appropriate protected iodobenzenesulfonamides
7c and 7d, which were obtained from the known compounds
6c12 and 6d13 (Scheme 2). The nucleosides were converted to the
corresponding phosphoramidites 10 and 11 in reasonable yields.
The identity of the compounds was fully ascertained by NMR
(1H, 13C, 31P, COSY and/or HSQC) and HRMS.
The phosphoramidites 4,4 5, 10 and 11 were succesfully incor-
porated into oligodeoxynucleotides using an automated DNA

P. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 3843–3849
3845
Table 2
Hybridisation data for DNA:RNA duplexesa
Tm (D
Tm/mod.)b (°C)
B =
W
X
Y
Z
ON1
ON2
ON3
ON4
50-dGTG TBT TGC
50-dGTG TBB TGC
50-dGTG BBB TGC
50-dGTG BBB BGC
30.0c
(ꢀ1.0)
39.0c
(+4.0)
46.0c
(+5.0)
55.5c
(+6.1)
27.5
27.0
28.0
(ꢀ3.5)
(ꢀ4.0)
(ꢀ3.0)
36.5
30.5
30.0
(+2.8)
45.5
(+4.8)
56.0
(ꢀ0.2)
34.5
(+1.2)
42.0
(ꢀ0.5)
34.0
(+1.0)
42.5
(+6.2)
(+2.7)
(+2.9)
Mismatch sequencesd
ON4
50-dGTG BBB BGC
30-rCAC ACA ACG
50-dGTG BBB BGC
30-rCAC AGA ACG
50-dGTG BBB BGC
30-rCAC AUA ACG
33.5
33.0
27.0
22.0
(ꢀ22.0)
(ꢀ23.0)
(ꢀ15.0)
(ꢀ20.5)
ON4
ON4
46.0c
46.5
33.0
30.0
(ꢀ9.5)
36.5
(ꢀ19.0)
(ꢀ9.5)
37.0
(ꢀ19.0)
(ꢀ9.0)
22.0
(ꢀ20.0)
(ꢀ12.5)
20.0
(ꢀ22.5)
a
b
c
Matched target sequence 50-rGCA AAA CAC.
See Table 1. For the unmodified reference duplex, Tm = 31.0 °C.
Data taken from Ref. 4
d
Mismatch studies, in brackets the changes in melting temperature as compared to the matched duplex ON4:RNA.
(a) DNA:DNA duplex
(b) DNA:RNA duplex
slightly increased in the case of both isomers W (
D
Tm = ꢁ+0.7 °C
per mod.) and X (
D
Tm = ꢁ+0.9 °C per mod.). In the case of alkynyl
monomers Y and Z, three or four consecutive incorporations virtu-
ally fail to evoke any further response, stabilizing or destabilizing.
However, the meta isomer Z seems to be slightly more destabiliz-
ing than the para isomer Y. Figure 1a shows a graphic connection
4
-1
-6
4
-1
-6
W
X
Y
Z
W
X
Y
Z
0
1
2
3
4
0
1
2
3
4
between
DTm and the number of modified moieties in the DNA:D-
NA duplex.
Number of modifications
Number of modifications
Table 2 shows the hybridisation data of the modified DNA:RNA
duplexes. A single incorporation of either W, X, Y or Z in ON1 leads
to decreases in Tm that were somewhat smaller than with DNA:D-
NA in case of triazolyl monomers W and X, whereas the decrease in
Tm was larger than with DNA:DNA in case of alkynylnucleoside
monomers Y and Z. Surprisingly, a larger decrease in Tm (ꢀ3.5 °C)
was observed with the meta-sulfonamido isomer of phenyltriazol-
ylnucleoside monomer X while a smaller decrease in Tm (ꢀ1 °C)
was observed with the para-sulfonamido isomer W. Incorporation
of the second modified nucleoside in ON2 leads to dramatically en-
hanced duplex stabilities in case of both triazolylnucleosides W
and X, although the effect of the new meta isomer X is slightly
Figure 1. Graphical illustration of the hybridisation versus the number of
modifications.
2.2. Hybridisation studies
The standard sequence chosen for the present study was a 9-
mer oligonucleotide sequence studied previously.3,4 with 1–4 con-
secutive incorporations of W, X, Y and Z. The oligonucleotides were
mixed in medium salt buffer with the complementary DNA and
RNA sequences and the melting temperatures (Tm) of the resulting
duplexes were derived from the UV melting curves at neutral pH 7.
Table 1 shows the results obtained with modified DNA:DNA du-
plexes. A single incorporation of either of the modified monomers
W, X, Y or Z in the centre of the duplex, ON1, lead to a significant
decrease in duplex stability. However, the decrease in thermal sta-
bility is less pronounced in the case of alkynyl monomers as vali-
smaller as compared to para isomer W (DTm = +4.0 °C and +2.8 °C
per modification, respectively). Overall, the introduction of the sec-
ond phenyltriazole moiety on top of the first gave an identical
increase in Tm of +9.0 °C in both cases (compare ON2 with ON1).
In comparison, the introduction of a second alkynylnucleoside
monomer Y and Z gave increases in Tm of +3.5 °C and +2.0 °C,
respectively, however, still maintains an overall slightly destabi-
lized duplex as compared to the unmodified duplex.
The tendency to stabilize the duplex with each successive incor-
poration continued in general by three and four incorporations in
ON3 and ON4 revealing further relative increases in duplex stabil-
dated by
D
Tm’s of around –1.5 °C compared to the unmodified
Tm’s
duplex in case of alkynyl monomers Y and Z as compared to
D
of around –5 °C in case of triazolyl monomers Y and Z. There was
absolutely no influence whatsoever with respect to the position
of sulfonamide group being para or meta. With the second incorpo-
ration of the modified monomers in ON2, the destabilizing influ-
ence in the case of triazolyl monomers W and X was reduced. In
fact, a relative increase in duplex stability was observed when
comparing ON2 with ON1. On the other hand, the destabilizing
influence of alkynyl monomers was found to be additive with
ity with
D
Tm’s of +6.1 °C per modification for the four sulfonamide
Tm’s of +2.8 °C per
substituted phenyltriazoles in W and X, and
D
modification for the four sulfonamide substituted phenylalkynyls
in Y and Z. In other words, the forth incorporation of a triazole
derivative increased the overall thermal stability of the duplex
with around 10 °C (comparing ON3 with ON4), whereas the forth
incorporation of an alkynyl derivative increased the overall ther-
mal stability of the duplex with around 7.5 °C. Overall, no differ-
ences whatsoever could be observed between meta and para
isomers concerning relative duplex stabilities with three or four
consecutive incorporations in ON3 and ON4. Figure 1b demon-
D
Tm’s of around ꢀ1.5 for each of the two incorporations of Y or
Z. In the case of triazolyl monomers W and X, the same trend
continued with the three incorporations in ON3, whereby the
destabilizing influence levelled off and the duplexes demonstrated
unchanged thermal stability as compared to the unmodified
duplex. With the four incorporations of W and X in ON4, the de-
crease in duplex stability was fully compensated by the stacking
of the modified nucleobases. Hence the duplex stabilities were
strates graphically how the
DTm evolves by the number of modifi-

3846
P. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 3843–3849
cations clearly showing the deep decreases by one modification
changing to large increases by consecutive incorporations.
the adjacent phenylgroups. Modelling has indicated an optimal
positioning of these in the triazoles.4,5
Mismatch studies were also performed in order to further probe
the RNA recognition. Hence, ON4 with either W, X, Y or Z was
mixed with RNA-sequences containing a single central mismatch,
and the melting temperatures of the mismatched duplex were
determined (Table 2). In all cases, fine mismatch discrimination
was observed as indicated by the large decreases in Tm relative
to the matched duplexes formed by ON4. A to C mismatches were
perfectly discriminated by all the four modified nucleosides W, X,
The very similar hybridisation properties of W and X demon-
strate that the efficient stacking is more dependent on the large
aromatic surfaces of the combined phenyl and triazole rings than
on the positioning of the sulfonamide. In other words, the organi-
sation of neighbouring sulfonamide group, indicated by modelling
on ON4 containing W,4 might be equally valid in the case of the
meta substituted rings in X. This also indicates a large room for
different substituents on the phenyltriazoles without compromis-
ing the high RNA-affinity.
Y and Z (DTm values >–15 °C). Similar results were observed for the
A to U mismatches, whereas the discrimination of the A to G
mismatches was slightly less efficient for all the four modifications
W–Z with the largest discrimination attained by the meta-sulfon-
In conclusion, phenyltriazole substituents on the 5-position of
uracil with or without sulfonamides is a simple very easily synthe-
sized tool for designing oligonucleotides with very high RNA-affin-
ity for potential antisense therapeutics. The demand for two
consecutive incorporations is complimented by the fact that also
a phenyltriazole on the 5-position of cytosine can participate in
the stacking and add to the increase in affinity.5 We hope that
these and other building blocks that are in progress can be signif-
icant tools in the development of therapeutic oligonucleotides.
amidophenyl alkynylnucleoside monomer
Z (DTm = –12.5 °C).
Overall, this is similar to unmodified DNA:RNA duplexes, where
the A to G mismatch is the most stable of the three.
3. Discussion
In the present study, we have investigated the directional influ-
ence of the sulfonamide moiety in complementing the aromatic
stacking in the major groove of nucleic acid duplexes. Hence, our
recent results indicated a contribution of the sulfonamide moiety
in improving the stacking efficiency of C5-phenyltriazolenucleo-
sides.4 At the same time, the study presents an opportunity to
directly compare the stacking efficiency of the triazole and ethynyl
linker in between the phenylsulfonamide and the nucleobase in
the modified nucleoside.
As far as the optimum number of consecutive incorporations of
modified C5-functionalized nucleosides is concerned, the hybrid-
isation data for the oligonucleotides of the current study validates
the trend of our first study demonstrating that one incorporation of
any of the four modified nucleoside monomers leads to decreased
stability of both DNA:DNA and DNA:RNA duplexes (Fig. 1). On the
other hand, four consecutive incorporations demonstrate a mas-
sive increase in stability of the DNA:RNA duplex while having a
net neutral effect on the stability of the DNA:DNA duplex. In the
case of phenyltriazoles, two consecutive incorporations are enough
to give most of the positive effect (Table 2 and Fig. 1). All the
modified nucleosides demonstrate potent preferential recognition
of RNA as compared to DNA while maintaining the capability of
mismatch discrimination. In future antisense applications, the
sequence design will be a balance between the RNA-affinity and
overall sequence specificity and might be optimal with two
consecutive incorporations.
Comparing the influence of substitution pattern in the phenyl-
triazole, the two isomeric sulfonamidonucleosides W and X exhib-
ited almost similar effect on duplex stability indicating virtually
non-existent influence of the position of sulfonamide on the phe-
nyl ring. Similarly, no obvious influence of substitution pattern of
sulfonamide group in C5-alkynylnucleosides was observed as there
was only a slight difference in duplex stability of two modified
nucleosides Y and Z.
A direct comparison of triazolyl and alkynyl nucleosides clearly
demonstrates that the stacking efficiency of triazole group is sig-
nificantly better than that of an alkynyl group (compare ONs hav-
ing W or X with Y or Z) attaining a maximum increase in duplex
stability of +6.1 °C per modification in case of C5-triazole modified
nucleosides as compared to +2.9 °C per modification in case of
C5-alkynyl modified nucleosides. This indicates that overall the tri-
azole is a much more efficient moiety for obtaining a high RNA-
affinity but also much more dependent on at least two consecutive
incorporations. This might be due to a combination of the in-
4. Conclusions
Three new simple building blocks have been synthesized from
the easily available 5-ethynyl-20-deoxyuridine introducing alkynyl
as well as triazole moieties into the major groove of nucleic acid
duplexes. The stacking of sulfonamide substituted phenyltriazoles
and phenylalkynyls in the major groove leads to very stable
DNA:RNA duplexes. However, significantly more stable duplexes
are attained with the C5-triazole modified nucleosides as
compared to the C5-alkynyl modified nucleosides, and only two
consecutive phenyltriazole moieties are necessary to give a pro-
nounced effect. Hereby, the new building blocks can be promising
tools in antisense research.
5. Experimental section
All commercial reagents were used as supplied, except CH2Cl2
which was distilled prior to use. Anhydrous solvents were dried
over 4 Å activated molecular sieves (CH2Cl2, pyridine and DCE) or
3 Å activated molecular sieves (CH3CN). Reactions were carried
out under argon or nitrogen when anhydrous solvents were used.
All mixtures of solvents were prepared as a volume to volume ratio
(v/v). All reactions were monitored using TLC analysis with Merck
silica gel plates (60 F254). To visualize the plates, they were exposed
to UV light (254 nm) and/or immersed in a solution of 5% H2SO4 in
methanol (v/v) followed by charring. Column chromatography was
performed with Silica Gel 60 (particle size 0.040–0.063 lm,
Merck). NMR spectra were recorded on a Varian Gemini 2000 spec-
trometer or on Bruker Advance III 400/300 spectrometers. Values
for d are in ppm relative to tetramethylsilane as an internal stan-
dard or 85% H3PO4 as an external standard (31P NMR). Assignments
of NMR-signals when given are based on 2D spectra and follow
standard nucleoside convention. High Resolution ESI mass spectra
were recorded on a Thermo Finnigan TSQ 700 spectrometer, and EI
mass spectra were recorded on a Finnigan SSQ 710 spectrometer.
IR spectra were recorded either on ABB MB 3000 DTGS or on Per-
kin–Elmer 1720 Infrared Fourier Transform spectrometer.
5.1. Synthesis of 5-(1-(3-(N-((dimethylamino)methylidene)
aminosulfonyl)phenyl)-1,2,3-triazol-4-yl)-50-(4,40-
dimethoxytrityl)-20-deoxyuridine (3)
To a solution of nucleoside 1 (300 mg, 0.53 mmol) and pro-
tected 3-azidobenzenesulfonamide 7b (210 mg, 0.83 mmol) in
creased surface for
p–p-stacking and the different orientation of

P. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 3843–3849
3847
THF:H2O:t-BuOH (10 mL, 3:1:1, v/v/v) was added aq sodium ascor-
bate (1 M, 1.2 mL, 1.2 mmol) and aq CuSO4 (1.1 mL, 7.5%, w/v,
0.32 mmol). The resulting clear solution was stirred at rt for 6 h,
and then diluted with EtOAc (30 mL) and brine (30 mL). The phases
were separated, and the organic phase was washed with a satu-
rated aqueous solution of NaHCO3 (30 mL). The combined aqueous
phase was extracted with EtOAc (2 ꢂ 30 mL), and the combined or-
ganic phase was dried (Na2SO4), and concentrated under reduced
pressure. The residue was purified by column chromatography
(0–5% MeOH in CH2Cl2) to afford the nucleoside 3 (225 mg, 51%)
as a pale yellow solid. Rf 0.4 (5% MeOH in CH2Cl2); 1H NMR (CDCl3,
400 MHz) d 9.86 (br s, 1H, NH), 8.87 (s, 1H, HC@N)), 8.59 (s, 1H, tri-
azole-H), 8.32 (s, 1H, Ar), 8.18 (s, 1H, H-6), 8.00 (d, 1H, J = 8.0 Hz,
Ar), 7.95 (d, 1H, J = 8.0 Hz, Ar), 7.60 (t, 1H, J = 8.0 Hz, Ar), 7.41 (d,
1H, J = 8.0 Hz, Ar), 7.31–7.34 (m, 4H, Ar), 7.23 (t, 2H, J = 8.0 Hz,
Ar), 7.11-7.15 (m, 2H, Ar), 6.80 (d, 2H, J = 8.0 Hz, Ar), 6.78 (d, 2H,
J = 8.0 Hz, Ar), 6.43 (t, 1H, J = 8.0 Hz, H-10), 4.44 (m, 1H, H-30),
3.72 (s, 6H, 2 ꢂ OCH3), 3.47 (dd, J = 10.2, 4.4 Hz, 1H, H-50), 3.41
(dd, J = 10.2, 4.4 Hz, 1H, H-50), 3.12 (s, 3H, CH3), 3.02 (s, 3H, CH3),
2.92 (d, ex, J = 8.0 Hz, 1H, 30-OH), 2.57 (m, 1H, H-20), 2.31–2.36
(m, 1H, H-20); 13C NMR (CDCl3, 100 MHz) d 161.3, 159.5, 158.4,
149.6, 144.6, 144.2, 139.9, 137.3, 136.8, 135.7, 135.6, 130.2,
130.1, 130.0, 128.1, 127.8, 126.8, 123.6, 120.2, 118.2, 113.2,
105.7, 86.8, 86.1, 85.8, 77.3, 77.0, 76.7, 72.4, 63.7, 55.2, 41.6,
40.6, 35.7. HiRes ESI MS m/z (M+Na) found/calcd 830.2554/
830.2584.
1H, H-6); 13C NMR (CDCl3/DMSO-d6, 100 MHz): d 145.6, 140.2,
130.0, 121.87, 121.86, 116.1.
5.4. Synthesis of N-dimethylaminomethylidine-3-
azidobenzenesulfonamide (7b)
To a cold stirred solution of POCl3 (0.48 mL, 5.05 mmol) in
dimethylformamide (10 mL) was added 3-azidobenzenesulfona-
mide 6b (500 mg, 2.52 mmol). The reaction mixture was stirred
at rt for 3 h, and then poured into cold water and neutralized with
saturated aqueous ammonia. The formed precipitates were iso-
lated, washed with water (50 mL) and dried to afford the product
7b (440 mg, 69%) as a white solid. Rf 0.3 (50% EtOAc in petroleum
ether). Mp 136–138 °C. IR (KBr) cmꢀ1 2106 (azide stretch), 1628
(C@N stretch), 1335 & 1119 (SO2 stretch). 1H NMR (DMSO-d6,
400 MHz): d 8.21 (s, 1H, CH@N), 7.58 (m, 1H, H-4), 7.51 (t, 1H,
J = 7.84 Hz, H-5), 7.45 (t, 1H, J = 1.84 Hz, H-2), 7.2 (m, 1H, H-6),
3.20 (s, 3H, NCH3), 2.99 (s, 3H, NCH3); 13C NMR (CDCl3/DMSO-d6,
100 MHz): d 159.6, 144.3, 140.1, 130.1, 122.2, 121.8, 116.3, 41.0
(NCH3), 35.0 (NCH3).
5.5. Synthesis of N-dimethylaminomethylidine-4-
iodobenzenesulfonamide (7c)
To a cold stirred solution of POCl3 (1.95 mL, 21.20 mmol) in
dimethylformamide (25 mL) was added 4-iodobenzenesulfona-
mide.12 6c (3.0 g, 10.60 mmol). The reaction mixture was stirred
at rt for 3 h, and then poured into cold water and neutralized with
saturated aqueous ammonia. The formed precipitates were iso-
lated, washed with water (200 mL) and dried to afford the product
7c (2.33 g, 65%) as a light orange solid. Rf 0.3 (50% EtOAc in petro-
leum ether). Mp 172–174 °C. IR (KBr) cmꢀ1 1620 (C@N stretch),
1142 & 1342 (SO2 stretch). 1H NMR (DMSO-d6, 400 MHz): d 8.20
(s, 1H, CH@N), 7.91 (d, 2H, J = 8.0 Hz, H-2 & 6), 7.53 (d, 2H,
5.2. Synthesis of 5-(1-(3-(N-((dimethylamino)methylidene)
aminosulfonyl)phenyl)-1,2,3-triazol-4-yl)-50-(4,40-
dimethoxytrityl)-30-O-(P-(2-cyanoethoxy)-N,N0-
diisopropylaminophosphinyl)-20-deoxyuridine (5)
The nucleoside 3 (135 mg, 0.16 mmol) was dried by the co-evap-
oration with anhydrous 1,2-dichloroethane (2 ꢂ 5 mL) and dis-
solved in anhydrous CH2Cl2 (5 mL). DIPEA (0.15 mL, 0.86 mmol)
and 2-cyanoethyl-N,N0-diisopropyl-phosphoramidochloridite (0.12
mL, 0.50 mmol) were added, and the reaction mixture was stirred
at rt for 2 h whereupon it was quenched with 2–3 drops of absolute
ethanol and evaporated to dryness. The resulting crude residue was
purified by column chromatography (0–2% MeOH in CH2Cl2) to af-
ford the phosphoramidite 5 (140 mg, 80%) as a white foam. Rf 0.4
(2% MeOH in CH2Cl2). 31P NMR (CDCl3, 162 MHz) d 149.1, 148.7.
HiRes ESI MS m/z (M+Na) found/calcd 1030.3617/1030.3663.
J = 8.0 Hz, H-3 & 5), 3.14 (s, 3H, NCH3), 2.90 (s, 3H, NCH3);13
C
NMR (DMSO-d6, 100 MHz) d 159.8, 142.6, 137.7, 127.6, 99.3, 40.8
(NCH3), 35.0 (NCH3).
5.6. Synthesis of N-dimethylaminomethylidine-3-
iodobenzenesulfonamide (7d)
To a cold stirred solution of POCl3 (0.82 mL, 8.83 mmol) in
dimethylformamide (15 mL) was added 3-iodobenzenesulfona-
mide.13 6d (1.25 g, 4.41 mmol). The reaction mixture was stirred
at rt for 3 h, and then poured into cold water and neutralized with
saturated aqueous ammonia. The formed precipitates were iso-
lated, washed with water (150 mL) and dried to afford the product
7d (1.22 g, 82%) as white solid. Rf 0.3 (50% EtOAc in petroleum
ether). Mp 140–143 °C. IR (KBr) cmꢀ1 1620 (C@N stretch), 1142
& 1335 (SO2 stretch). 1H NMR (DMSO-d6, 300 MHz) d 8.22 (s, 1H,
CH@N), 8.05 (s, 1H, H-2), 7.93 (d, 1H, J = 7.8 Hz, H-6), 7.78 (d, 2H,
J = 7.8 Hz, H-4), 7.33 (t, 1H, J = 7.8 Hz, H-5), 3.14 (s, 3H, NCH3),
2.90 (s, 3H, NCH3); 13C NMR (CDCl3/DMSO-d6, 100 MHz) d 159.5,
144.3, 140.0, 134.1, 130.3, 125.1, 93.8, 41.1 (NCH3), 35.1 (NCH3).
5.3. Synthesis of 3-azidobenzenesulfonamide (6b)
To an ice cold stirred solution of metanilamide11 (1.0 g,
5.81 mmol) in 4 N HCl (50 mL) was added an aqueous NaNO2 solu-
tion (0.48 g, 6.97 mmol in 3 mL H2O) dropwise, maintaining the
temperature of reaction mixture at 0–5 °C. To the diazotized mix-
ture was then added a saturated aqueous solution of NaHCO3 drop-
wise slowly to neutralize the reaction mixture (pHꢁ7.0), carefully
maintaining the temperature below 5 °C. To this neutralized solu-
tion was added rapidly aqueous NaN3 solution (1.13 g, 17.43 mmol
in 5 mL H2O) and the solution was stirred vigorously for 1 h. The
reaction mixture was extracted with ethyl acetate (3 ꢂ 20 mL).
The organic extract was washed with brine (2 ꢂ 20 mL), dried
(MgSO4) and concentrated under reduced pressure to afford the
target azide 6b (0.77 g, 67%) as an orange solid. The solid was
washed with 25 mL petroleum ether, dried and used without fur-
ther purification in the next step. Rf 0.6 (50% EtOAc in petroleum
ether). Mp 127–128 °C. IR (KBr) cmꢀ1 3340 & 3263 (N-H stretch),
2114 (azide stretch), 1319 & 1173 (SO2 stretch). 1H NMR (DMSO-
d6, 400 MHz): d 7.65 (m, 1H, H-4), 7.58 (t, 1H, J = 1.92 Hz, H-2),
7.51 (t, 1H, J = 7.96 Hz, H-5), 7.33 (s, ex, 2H, SO2NH2), 7.22 (m,
5.7. Synthesis of 5-(4-(N-((dimethylamino)methylidene)
aminosulfonyl)phenyl)ethynyl-50-(4,40-dimethoxytrityl)-20-
deoxyuridine (8)
The nucleoside 1 (550 mg, 1.0 mmol), protected 4-iodobenzene-
sulfonamide 7c (500 mg, 1.5 mmol), CuI (40 mg, 0.2 mmol) and
Pd(PPh3)4 (120 mg, 0.1 mmol) were suspended in anhydrous DMF
(10 mL) and the contents were degassed and placed under an argon
atmosphere while stirring in the dark for 30 min at rt. Anhydrous
Et3N (0.6 mL, 4.25 mmol) was added and the mixture was stirred

3848
P. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 3843–3849
overnight in the dark at rt under argon atmosphere. After reaction
completion, the mixture was concentrated under reduced pressure
and added EtOAc (70 mL) and saturated aqueous solution of NaH-
CO3 (50 mL). The phases were separated, and the organic phase
was washed with brine (50 mL). The combined aqueous phase
was extracted with EtOAc (40 mL), and the combined organic
phase was dried (Na2SO4), and concentrated under reduced pres-
sure to get the crude product which was purified by column chro-
matography (0–5% MeOH in CH2Cl2) to afford the nucleoside 8
(500 mg, 65%) as a white foam. Rf 0.4 (5% MeOH in CH2Cl2). 1H
NMR (CDCl3, 400 MHz) d 8.47 (br s, 1H, NH), 8.33 (s, 1H, HC@N),
8.09 (s, 1H, H-6), 7.61 (d, 2H, J = 8.0 Hz, Ar), 7.43–7.45 (m, 2H,
Ar), 7.31–7.34 (m, 4H, Ar), 7.24–7.28 (m, 2H, Ar), 7.14 (m, 1H,
Ar), 6.94 (d, 2H, J = 8.0 Hz, Ar), 6.77 (d, 2H, J = 8.0 Hz, Ar), 6.75 (d,
2H,, J = 8.0 Hz, Ar), 6.36 (t, 1H, J = 8.0 Hz, H-10), 4.57 (m, 1H, H-30),
4.12 (m, 1H, H-40), 3.69 (s, 6H, 2 ꢂ OCH3), 3.53 (dd, J = 10.2,
4.4 Hz, 1H, H-50), 3.29 (dd, J = 10.2, 4.4 Hz, 1H, H-50), 3.13 (s, 3H,
CH3), 3.02 (s, 3H, CH3), 2.53 (m, 1H, H-20), 2.35 (m, 1H, H-20),
2.16 (br s, 1H, 30-OH); 13C NMR (CDCl3, 100 MHz) d 161.4, 159.3,
158.8, 149.3, 144.5, 143.0, 142.4, 135.6, 135.5, 131.8, 130.0,
128.3, 128.2, 128.0, 127.2, 126.3, 126.1, 113.5, 100.1, 92.5, 87.2,
86.8, 85.9, 83.0, 72.3, 63.5, 55.3, 41.9, 41.6, 35.7. HiRes ESI MS m/
z (M+Na) found/calcd 787.2398/787.2408.
and 2-cyanoethyl-N,N0-diisopropylphosphoramidochloridite (0.20
mL, 0.84 mmol) were added, and the reaction mixture was stirred
at rt for 2 h whereupon it was quenched with 2–3 drops of absolute
ethanol and evaporated to dryness. The resulting crude residue
was purified by column chromatography (0–2.5% MeOH in CH2Cl2)
to afford the phosphoramidite 10 (170 mg, 67%) as a white foam. Rf
0.4 (2% MeOH in CH2Cl2). 31P NMR (CDCl3, 162 MHz) d 149.1, 148.6.
HiRes ESI MS m/z (M+Na) found/calcd 987.3514/987.3492.
5.10. Synthesis of 5-(3-(N-((dimethylamino)methylidene)
aminosulfonyl)phenyl)ethynyl-50-(4,40-dimethoxytrityl)-30-O-
(P-(2-cyanoethoxy)-N,N0-diisopropylaminophosphinyl)-20-
deoxyuridine (11)
The nucleoside 9 (200 mg, 0.26 mmol) was dried by the co-
evaporation with anhydrous 1,2-dichloroethane (2 ꢂ 5 mL) and
dissolved in anhydrous CH2Cl2 (5 mL). DIPEA (0.30 mL, 1.72 mmol)
and 2-cyanoethyl-N,N0-diisopropylphosphoramidochloridite (0.20
mL, 0.84 mmol) were added, and the reaction mixture was stirred
at rt for 2 h whereupon it was quenched with 2–3 drops of absolute
ethanol and evaporated to dryness. The resulting crude residue
was purified by column chromatography (0–2.5% MeOH in CH2Cl2)
to afford the phosphoramidite 11 (185 mg, 73%) as a white foam. Rf
0.4 (2% MeOH in CH2Cl2). 31P NMR (CDCl3, 162 MHz) d 149.0, 148.6.
HiRes ESI MS m/z (M+Na) found/calcd 987.3453/987.3492.
5.8. Synthesis of 5- (3-(N-((dimethylamino)methylidene)
aminosulfonyl)phenyl)ethynyl-50-(4,40-dimethoxytrityl)-20-
deoxyuridine (9)
5.11. Synthesis of oligodeoxynucleotides
Oligonucleotide synthesis was carried out on an automated
DNA synthesizer following the phosphoramidite approach. Synthe-
sis of oligonucleotides ON1–ON4 (W–Z) was performed on a
The nucleoside 1 (550 mg, 1.0 mmol), protected 3-iodobenzene-
sulfonamide 7d (500 mg, 1.5 mmol), CuI (40 mg, 0.2 mmol) and
Pd(PPh3)4 (120 mg, 0.1 mmol) were suspended in anhydrous DMF
(10 mL) and the mixture was degassed and placed under an argon
atmosphere while stirring in the dark for 30 min at rt. Anhydrous
Et3N (0.6 mL, 4.25 mmol) was added and the mixture was stirred
overnight in the dark at rt under argon atmosphere. After reaction
completion, the mixture was concentrated under reduced pressure
and added EtOAc (70 mL) and saturated aqueous solution of NaHCO3
(50 mL). The phases were separated, and the organic phase was
washed with brine (50 mL). The combined aqueous phase was ex-
tracted with EtOAc (40 mL), and the combined organic phase was
dried (Na2SO4), and concentrated under reduced pressure to get
the crude product which was purified by column chromatography
(0–5% MeOH in CH2Cl2) to afford the nucleoside 9 (500 mg, 65%)
as a white foam. Rf 0.4 (5% MeOH in CH2Cl2). 1H NMR (CDCl3,
400 MHz) d 9.17 (br s, 1H, NH), 8.23 (s, 1H, HC@N), 8.08 (s, 1H, H-
6), 7.77–7.80 (m, 2H, Ar), 7.42–7.43 (m, 2H, Ar), 7.32–7.35 (m, 4H,
Ar), 7.18–7.26 (m, 4H, Ar), 7.14 (m, 1H, Ar), 6.78 (d, 2H, J = 8.0 Hz,
Ar), 6.76 (d, 2H, J = 8.0 Hz, Ar), 6.36 (t, 1H, J = 8.0 Hz, H-10), 4.58
(m, 1H, H-30), 4.14 (m, 1H, H-40), 3.67 (s, 6H, 2 ꢂ OCH3), 3.43 (dd,
J = 10.2, 4.4 Hz, 1H, H-50), 3.35 (dd, J = 10.2, 4.4 Hz, 1H, H-50), 3.13
(s, 3H, CH3), 2.96 (s, 3H, CH3), 2.92 (br s, 1H, 30-OH), 2.7 (m, 1H, H-
20), 2.34 (m, 1H, H-20); 13C NMR (CDCl3, 100 MHz) d 161.3, 159.3,
158.6, 149.1, 144.4, 142.8, 142.4, 135.5, 129.98, 129.91, 129.3,
128.3, 128.0, 127.8, 127.0, 123.3, 113.3, 99.9, 92.2, 87.1, 86.7, 86.0,
81.5, 72.3, 63.5, 55.2, 41.7, 41.5, 35.5. HiRes ESI MS m/z (M+Na)
found/calcd 787.2382/787.2408.
0.2 lmol scale by using the amidites 4, 5, 10 and 11 as well as
the corresponding commercial 2-cyanoethyl phosphoramidites of
the natural 20-deoxynucleosides. The synthesis followed the regu-
lar protocol for the DNA synthesizer. For compound 4, 5, 10 and 11,
a prolonged coupling time of 15 min was used. 1H-Tetrazole was
used as the activator and coupling yields for all 2-cyanoethyl phos-
phoramidites were 95–99.8%. The 50-O-DMT-ON oligonucleotides
were removed from the solid support by treatment with concen-
trated aqueous ammonia at 55 °C for 16 h, which also removed
the protecting groups. The oligonucleotides were purified by re-
versed-phase HPLC on a Waters 600 system using a Xterra prep
MS C18; 10
l
m; 7.8ꢂ150 mm column; Buffer A: 0.05 M Triethyl
Ammonium Acetate pH 7.4. Buffer B: MeCN/H2O (1:1). Program
used: 2 min 100% A, 100–30% A over 38 min, 10 min 100% B,
10 min 100% A. All oligonucleotides were detritylated by treatment
with an 80% aqueous solution of acetic acid for 30 min, quenched
with an aqueous solution of sodium acetate (3 M, 15
lL) and then
added sodium perchlorate (5 M, 15 L) followed by acetone (1 mL).
l
The pure oligonucleotides precipitated over night at ꢀ20 °C. After
centrifugation 12000 rpm, 10 min at 4 °C, the supernatant was re-
moved, and the pellet washed with cold acetone (2 ꢂ 1 mL) and
dried for 30 min under reduced pressure, and dissolved in pure
water (1000 lL). The concentration was determined by UV at
260 nm, and the purity confirmed by IC analysis. MALDI-TOF-MS
[MꢀH]ꢀ gave the following results (calcd/found): ON1-X (2943.6/
2941.3); ON2-X (3151.4/3151.1); ON3-X (3359.2/3361.4); ON4-X
(3567.0/3570.9); ON1-Y (2900.6/2900.9); ON2-Y (3065.4/3065.8);
ON3-Y (3230.2/3229.8); ON4-Y (3395.0/3398.4); ON1-Z (2900.6/
2901.6); ON2-Z (3065.4/3067.6); ON3-Z (3230.2/3231.5); ON4-Z
(3395.0/3397.4);
5.9. Synthesis of 5-(4-(N-((dimethylamino)methylidene)
aminosulfonyl)phenyl)ethynyl-50-(4,40-dimethoxytrityl)-30-O-
(P-(2-cyanoethoxy)-N,N0-diisopropylaminophosphinyl)-20-
deoxyuridine (10)
6. Thermal denaturation experiments
The nucleoside 8 (200 mg, 0.26 mmol) was dried by the co-
evaporation with anhydrous 1,2-dichloroethane (2 ꢂ 5 mL) and
dissolved in anhydrous CH2Cl2 (5 mL). DIPEA (0.30 mL, 1.72 mmol)
UV melting experiments were carried out on a UV spectrometer.
Samples were dissolved in a medium salt buffer containing 2.5 mM

P. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 3843–3849
3849
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ˇ
3. Kocalka, P.; Andersen, N. K.; Jensen, F.; Nielsen, P. ChemBioChem 2007, 8, 2106–
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l
sequences. The increase in absorbance at 260 nm as a function of
time was recorded while the temperature was increased linearly
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ature programmer. The melting temperature was determined as the
local maximum of the first derivatives of the absorbance vs. temper-
ature curve. The melting curves were found to be reversible. All
determinations are averages of at least duplicates within 0.5 °C.
4. Andersen, N. K.; Chandak, N.; Brulíková, L.; Kumar, P.; Jensen, M. D.; Jensen, F.;
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Acknowledgements
The project was supported by The Danish National Research
Foundation, The Danish Council for Independent Research | Natural
Sciences (FNU) and Technology and Production Sciences (FTP) and
Villum Kann Rasmussen Fonden. One of the authors (N.C.) is thank-
ful to the Council of Scientific and Industrial Research (CSIR), New
Delhi, India for the award of Senior Research Fellowship.
11. Kumler, W. D.; Halverstadt, I. F. J. Am. Chem. Soc. 1941, 63, 2182.
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References and notes
1. Zamecnik, P. C.; Stephenson, M. L. Proc. Nati. Acad. Sci. U.S.A. 1978, 75, 280–284.