Efficient RNA-targeting by the introduction of aromatic stacking in the duplex major groove via 5-(1-phenyl-1,2,3-triazol-4-yl)-2′-deoxyuridines

Article abstract of DOI:10.1016/j.bmc.2010.05.019

Three pyrimidine nucleosides with differently substituted phenyltriazoles attached to the 5-position were prepared by Cu(I)-assisted azide-alkyne cycloadditions (CuAAC) and incorporated into oligonucleotides. Efficient π-π-stacking between two or more phenyltriazoles in the major groove was found to increase the thermal stability of a DNA:RNA duplex significantly. The best stacking, and most stable duplex, was obtained by a sulfonamide substituted derivative.

Full text of DOI:10.1016/j.bmc.2010.05.019

Bioorganic & Medicinal Chemistry 18 (2010) 4702–4710
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Efficient RNA-targeting by the introduction of aromatic stacking in the
duplex major groove via 5-(1-phenyl-1,2,3-triazol-4-yl)-20-deoxyuridines
Nicolai Krog Andersen a, Navneet Chandak b, Lucie Brulíková a, , Pawan Kumar b, Michael Dalager Jensen a,
a,
Frank Jensen c, Pawan K. Sharma b, , Poul Nielsen
*
*
a Nucleic Acid Center,à Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense M, Denmark
b Department of Chemistry, Kurukshetra University, Kurukshetra 136 119, India
c Department of Chemistry, Aarhus University, Langelandsgade, 8000 Aarhus, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 January 2010
Revised 5 May 2010
Accepted 6 May 2010
Available online 12 May 2010
Three pyrimidine nucleosides with differently substituted phenyltriazoles attached to the 5-position
were prepared by Cu(I)-assisted azide–alkyne cycloadditions (CuAAC) and incorporated into oligonucle-
otides. 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. The best stacking, and most stable
duplex, was obtained by a sulfonamide substituted derivative.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Oligonucleotides
p–p-Stacking
Click chemistry
RNA-targeting
1. Introduction
(CuAAC)8 performed on 5-ethynyl-20-deoxyuridine (1, Scheme 1).9 This
building block has been used for Click Chemistry conjugation of various
The nucleic acid duplex constitutes an excellent scaffold for
chemically designed supramolecular chemistry.1 The duplex is
formed between complementary oligonucleotide sequences on
moieties to DNA10,11 and leads to the positioning of a triazole in the ma-
jor groove of the duplex. We found that one triazole, either unsubstitut-
ed or substituted with a phenyl or a benzyl group, in general leads to
decreased duplex stability, whereas four consecutive incorporations
lead to significant duplex stabilisation of a DNA:RNA duplex.9 Hence,
a 9-mer duplex with four triazoles in the centre (replacing the 5-methyl
groups of the bold thymidines in the duplex 50-dGTGTTTTGC:30-rCA-
CAAAACG) displayed an increase in melting temperature of 14 °C as
compared to the unmodified duplex, and a further 7 °C increase was
obtained by phenyl substituted triazoles (X in Scheme 1). Modelling
demonstrated (1) a clear preference for a coplanar orientation between
the pyrimidine and the triazole with the C5 of the triazole oriented to-
wards the O4 of the uracil (via a C–HꢀꢀꢀO interaction), and (2) a signif-
the basis of selective hydrogen-bonding and strong
p–p-stacking
of the nucleobases. With the aim of targeting RNA-sequences by
synthetic oligonucleotides, following the so-called antisense ap-
proach,2 the idea of synthetically increasing the stability of the du-
plex by increasing the stacking has been approached in several
ways including synthetic nucleobases with larger ring systems.3
A major example is a tricyclic phenoxazine replacing a cytosine
and increasing the thermal stability of a DNA:RNA duplex with
up to 5 °C per modification.4 Furthermore, the 5-position of pyrim-
idine nucleosides has been functionalised with the propyn-1-yl
group5 as well as with five-membered heterocycles,6 and these
modifications have been found to increase the duplex stability
icant intrastrand
p–p-stacking between the triazoles in the duplex
involving to some degree also the phenyl groups. In combination with
via increased p–p-stacking.
CD-spectroscopy, it was also shown that the duplexes are driven to-
In our former study, we followed the concept of Click Chemis-
wards A- or A/B-type like duplexes by the introduction of the
stacking triazoles.9
p–p-
try7 and studied the Cu(I)-catalysed azide–alkyne cycloadditions
In the present study, we explore the scope of this stacking effect
of phenyltriazoles concerning (1) the degree of modification in the
duplex necessary to obtain the stabilisation, and (2) the effect of
hydrophilic substituents in combination with electron-donating
and withdrawing properties. We therefore decided to introduce a
phenol and a sulfonamide (Y and Z, respectively, Scheme 1). To
our best knowledge, this is at the same time the first introduction
à
Nucleic Acid Center is funded by the Danish National Research Foundation for
studies on nucleic acid chemical biology.
* Corresponding authors. Tel.: +45 6550 2565; fax: +45 6615 8780 (P.N.).
E-mail address: pon@ifk.sdu.dk (P. Nielsen).
Present address: Department of Organic Chemistry, Faculty of Science, Palacky´
ˇ
University, Tr. 17. Listopadu 1192/12, Olomouc 771 46, Czech Republic.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.019

N. K. Andersen et al. / Bioorg. Med. Chem. 18 (2010) 4702–4710
4703
R'
by direct silylation of the known p-azidophenol 9, which has been
prepared from p-bromophenol.12 The TBDMS-group was chosen as
an appropriately base-sensitive protection for the phenol
group. For introducing a sulfonamide moiety, the known p-azido-
benzensulfonamide 11, made by a diazotation of p-aminobenzen-
sulfonamide,13 was protected with the equally base-sensitive
dimethylamidine group to give 12. The azides were reacted with
4 in the Cu(I)-catalysed cycloaddition to give the two protected
triazole nucleosides 5 and 6 in good yields (Scheme 1). These were
N
N
O
N
O
N
N
NH
NH
O
O
RO
RO
a,b or c
d or e
converted to the corresponding phophoramidites
respectively.
7 and 8,
O
O
The phosphoramidites 3, 7 and 8 were successfully incorpo-
rated into oligodeoxynucleotides using automated solid phase syn-
thesis with tetrazole as the activator and extended coupling times
for the modified phosphoramidites. After completion of the syn-
thesis, the oligonucleotides were removed from the solid support
by treatment with concentrated aqueous ammonia. This treatment
also removed all protecting groups including the silyl protection of
the phenol and the amidine protection of the sulfonamide giving
the incorporated monomers Y and Z, respectively, (Scheme 1).
The three monomers X, Y and Z were incorporated into the same
series of 9-mer oligonucleotides ON1–ON6 (Tables 1 and 2). The
constitution and purity of these were controlled by MALDI-MS
and RP-HPLC, respectively.
OH
OH
R = R' = H
2
1
R = H
4 R = DMT
5 R = DMT, R' = OTBDMS
6
R = DMT, R' = SO2N=CHN(CH3)2
R
R
N
N
N
N
O
N
O
N
N
NH
NH
O
N
O
2.2. Hybridisation studies
DMTrO
O
f
O
O
The series of oligonucleotides for this study was chosen with
the purpose of finding the minimum of modification needed for
duplex stabilisation. Hence, the 9-mer oligonucleotides with one
and four incorporations of X in the centre, ON1 and ON6, were ta-
ken from our former study,9 whereas ON2–ON5 represent different
positions of a single modification as well as two or three consecu-
tive incorporations of X. The same series ON1–ON6 was hereafter
prepared with both Y and Z (Table 1).
The hybridisation studies of the oligonucleotides were per-
formed by UV-spectroscopy. For determining concentrations,
extinction coefficients for oligonucleotides were determined by
standard methods using extinction coefficients for the single
nucleotides. For the modified monomers X and Y, the extinction
coefficients at 260 nm for the deprotected phenyl9 and hydroxy-
phenyltriazole nucleosides were determined by UV-measure-
O
P
O
P
O
O
(i-Pr)2N
O(CH2)2CN
3
X
R = H
R = H
Y R = OH
Z R = SO2NH2
7 R = OTBDMS
8 R = SO2N=CHN(CH3)2
Scheme 1. Reagents: (a) Ref. 9: PhBr, NaN3, CuI, Na ascorbate, EtOH, H2O, MW, 2
68%; (b) azide 10, CuI, Na ascorbate, pyridine, EtOH, H2O, 5 78%; (c) azide 12, CuSO4,
Na ascorbate, t-BuOH, H2O, THF, pyridine, 6 88%; (d) Ref. 9: (i) DMT–Cl, pyridine,
CH3CN; (ii) NC(CH2)2OPClN(iPr)2, EtN(iPr)2, CH2Cl2, 3 42%; (e) NC(CH2)2OPClN(iPr)2,
EtN(iPr)2, CH2Cl2,
dimethoxytrityl, TBDMS = t-butyldimethylsilyl.
7 74%, 8
67%; (f) automated DNA synthesis. DMT = 4,40-
of a sulfonamide into DNA. Ultimately, these aromatic nucleosides
are extremely simple building blocks for the design of oligonucle-
otides with improved and selective hybridisation to complemen-
tary RNA and hereby an interesting potential in antisense
therapeutics.2
ments. Ab initio calculations of UV-spectra for
a series of
derivatives of X indicated no increased absorption at 260 nm for
Z compared to X and therefore a similar extinction coefficient
was assumed. In order to secure a concentration of 1.5 lM for
the duplexes, however, a practical 50% lower coefficient for mono-
2. Results and discussion
2.1. Chemical synthesis
mer Z was applied in the hybridisation experiments.
The oligonucleotides were mixed with the complementary DNA
and RNA-sequences and the melting temperatures (Tm) of the
resulting duplexes were determined. Table 1 shows the results
In our first study, we prepared the triazole containing pyrimi-
dine nucleosides directly from the unprotected 5-ethynyl-20-deox-
yuridine 1 using sodium azide, aryl or alkyl halides, and an in situ
azidation/cycloaddition protocol (Scheme 1).9 Each nucleoside,
including 2, was hereafter protected at the 50-position by the
4,40-dimethoxytrityl (DMT) group and converted to 30-O-phospho-
ramidites in order to afford building blocks suitable for incorpora-
tion into oligonucleotides using standard automated solid phase
DNA synthesis. By the phosphoramidite 3, oligonucleotides con-
taining the 5-(1-phenyl-1,2,3-triazol-4-yl)-20-deoxyuridine moiety
X were obtained.9 For the present study, we obtained the best re-
sults by performing the cycloaddition reactions on the 50-O-DMT-
protected 5-ethynyl-20-deoxyuridine 4 using azides that were pre-
pared and isolated from suitable building blocks (Scheme 2). For
introducing a phenol moiety, the protected azide 10 was prepared
N
NH2
N
S
OH
OTBDMS
O
S
O
O
O
a
b
N3
N3
N3
N3
9
10
11
12
Scheme 2. Reagents: (a) TBDMS–Cl, DMAP, pyridine, CH3CN, 90%; (b) DMF, POCl3,
73%.

4704
N. K. Andersen et al. / Bioorg. Med. Chem. 18 (2010) 4702–4710
Table 1
monomers, ON6, the decrease in duplex stability was fully com-
pensated by the stacking of the modified nucleobases. Hence, the
duplex stabilities were similar to the unmodified duplex, varying
Hybridisation data for DNA:DNA duplexesa
Tm
(
D
Tm/mod.)/(°C)b
from a small decrease with the phenol Y (
mod.) to a small increase with the sulfonamide Z (DTm = +0.7 °C
per mod.).
Table 2 shows the hybridisation data of the modified DNA:R-
NA duplexes. A single incorporation of either X, Y or Z, in ON1
or ON2, lead to decreases in thermal stability that were some-
D
Tm = ꢁ1.0 °C per
B = X
Y
Z
ON1
ON2
ON3
ON4
ON5
ON6
50-dGTG TBT TGC
50-dGTG BTT TGC
50-dGTG BBT TGC
50-dGTG TBB TGC
50-dGTG BBB TGC
50-dGTG BBB BGC
28.0c
(ꢁ5.0)
30.0
28.5
28.0
(ꢁ4.5)
(ꢁ5.0)
29.5
29.0
(ꢁ3.0)
(ꢁ3.5)
29.0
(ꢁ4.0)
31.5
30.0
(ꢁ1.5)
30.0
(ꢁ2.0)
(ꢁ0.8)
what smaller than with DNA:DNA (
D
Tm’s between ꢁ0.5 and
28.5
30.5
ꢁ2.0 °C). This picture changed significantly by the introduction
of the second modified nucleoside. ON3 displayed significantly
increased duplex stabilities, and in the slightly different se-
(ꢁ1.5)
(ꢁ2.3)
29.0
(ꢁ1.3)
33.0
(0.0)
35.5
(+0.7)
30.0
(ꢁ1.0)
32.0c
(ꢁ0.3)
(ꢁ1.3)
29.0
quence ON4, even further increases were observed with
up to +4 °C per modification. The tendency continued by three
and four incorporations in ON5 and ON6 revealing further rela-
DTm’s
(ꢁ1.0)
a
Target sequence 50-dGCA AAA CAC.
b
Melting temperatures (Tm values/°C) obtained from the maxima of the first
tive increases in duplex stability with
DTm’s up to +6.1 °C per
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).
modification for the four sulfonamide substituted phenyltriazoles
Z. Interestingly, the hyperchromicity observed for the melting of
a duplex generally decreased by the numbers of monomer Z but
not in the case of monomers X and Y. Nevertheless, the melting
transitions were clearly determined.
lM
B (D
c
Data taken from Ref. 9.
Comparing the data for the modified DNA:RNA duplexes in a
different way, the introduction of the second phenyltriazole moi-
ety on the top of the first gave an increase in Tm of 5 °C (compare
ON3 with ON2, Table 2) or even 8.5 °C (compare ON4 with ON1)
for X. The third incorporation of X gave an increase in Tm of 5.5
or 8 °C (compare ON5 with ON3 or ON4), and the fourth incorpora-
tion of X gave a further increase in Tm of 8.5 °C (compare ON6 with
ON5). The corresponding increases in Tm for the phenol moiety Y
were +8/8.5° for the second, +6/+8 °C for the third and +6 °C for
the fourth incorporation. For the sulfonamide Z, the increases in
Tm were +6.5/9° for the second, +7/+10.5 °C for the third and
+9.5 °C for the fourth incorporation.
The remarkable RNA recognition was further investigated by
mismatch studies. Hence, ON6 with either X, Y or Z was mixed
with RNA-sequences containing a single central mismatch, and
the melting temperatures of the mismatched duplexes were deter-
mined (Table 2). In most cases, fine mismatch discrimination was
observed as indicated by the large decreases in Tm relative to the
matched duplexes formed by ON6. A to C mismatches were per-
Table 2
Hybridisation data for DNA:RNA duplexesa
Tm
(
D
Tm/mod.)/°Cb
B = X
Y
Z
ON1
ON2
ON3
ON4
ON5
ON6
50-dGTG TBT TGC
50-dGTG BTT TGC
50-dGTG BBT TGC
50-dGTG TBB TGC
50-dGTG BBB TGC
50-dGTG BBB BGC
29.0c
(ꢁ2.0)
30.0
30.5
30.0
(ꢁ0.5)
(ꢁ1.0)
29.0
29.0
(ꢁ1.0)
(ꢁ2.0)
37.0
(+3.0)
39.0
(+4.0)
45.0
(+4.7)
51.0
(ꢁ2.0)
35.5
(+2.3)
39.0
(+4.0)
46.0
(+5.0)
55.5
35.0
(+2.0)
37.5
(+3.3)
43.0
(+4.0)
51.5c
(+5.1)
(+5.0)
(+6.1)
Mismatch sequencesd
ON6
ON6
ON6
50-dGTG BBB BGC
24.0c
26.5
nte
30-rCAC ACA ACG
50-dGTG BBB BGC
30-rCAC AGA ACG
50-dGTG BBB BGC
30-rCAC AUA ACG
(ꢁ27.5)
(ꢁ24.5)
42.0c
41.0
46.0
(ꢁ9.5)
nte
fectly discriminated by X and Y (
D
Tm values of ꢁ27.5 and
(ꢁ9.5)
31.0c
(ꢁ20.5)
(ꢁ10.0)
29.0
(ꢁ22.0)
ꢁ24.5 °C, respectively), whereas no mismatched duplex by Z could
be detected. Similar results were observed for the A to U mis-
matches, whereas the discrimination of the A to G mismatches
a
Matched target sequence 50-rGCA AAA CAC.
Melting temperatures (Tm values/°C) obtained from the maxima of the first
was slightly smaller for all the three modifications X–Z (DTm’s
b
around ꢁ10 °C). This is however similar to unmodified DNA:RNA
duplexes, where the A to G mismatch is the most stable of the
three.
The fact that two mismatched duplexes formed by ON6-Z were
not detectable by UV-spectroscopy was puzzling but seemed asso-
ciated with the generally low hyperchromicity observed for the
DNA:RNA duplexes with Z. With the local denaturation induced
in the middle of the duplex by a mismatch the transition might
be undetectable by the UV-methodology.
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 = 31.0 °C).
lM
B (D
c
Data taken from Ref. 9.
Mismatch studies, in brackets the changes in melting temperature as compared
d
to the matched duplex ON6:RNA.
e
No transition observed.
obtained with modified DNA:DNA duplexes. A single incorporation
of either of the modified monomers X, Y or Z once in the centre of
the duplex, ON1, lead to a significant decrease in duplex stability as
validated by Tm’s around 5 °C lower than for the unmodified du-
plex. The same was observed for ON2 although the decreases in
Tm were somewhat smaller. With the second incorporation of the
modified monomers, in ON3 and ON4, the relative destabilisation
was even less pronounced, especially in the case of the sulfon-
2.3. Circular dichroism spectroscopy
To further study the influence of p–p-stacking and the stepwise
increasing modification on the duplex structure, circular dichroism
(CD) spectroscopy was applied. It is well known that DNA:DNA du-
plexes adopt a B-type form in solution, whereas RNA:RNA duplexes
adopt an A-type and DNA:RNA duplexes intermediate A/B-type
structures. A- and B-type duplexes are known to display distinctly
different CD spectra. A-type duplexes give an intense negative
band at ꢂ210 nm and a positive band at ꢂ260 nm, whereas B-type
amide Z (
D
Tm = ꢁ0.8 °C per mod.). The same trend continued with
three incorporations, ON5, and with four modified nucleoside

N. K. Andersen et al. / Bioorg. Med. Chem. 18 (2010) 4702–4710
4705
duplexes give a negative band at ꢂ250 nm and positive bands at
ꢂ220 and ꢂ280 nm. For this study the unmodified DNA:DNA du-
plex was taken as a standard for the B-type, and the CD-spectrum
(Fig. 1) clearly displayed the B-type characteristics. The DNA:RNA
duplex showed the expected intermediate A/B-type with some
clear A-type characteristics. The single incorporation of monomer
X in ON2 indicated that the DNA:DNA duplex retained its inherent
B-type form (Fig. 1). This is similar to what was observed for ON1-
X in our first study.9 However, the double incorporation of X in
ON3 and ON4 resulted in two slightly different CD-curves. ON3
resembled ON2, whereas ON4 showed a small shift of the band
at 280 nm towards 275 nm. Upon the triple incorporation of X in
ON5 we observed a further small shift towards the A/B-type helical
form with a beginning shoulder at 265 nm and a decreasing nega-
tive band at 250 nm. This is fully in accordance with our previous
observations for ON6-X.9
For the DNA:RNA hybrid duplex, only small changes in the CD-
spectra were observed by the introduction of X (Fig. 2). Hence, all
curves were similar to the one obtained from the unmodified
DNA:RNA duplex indicating that the modifications are not chang-
ing the overall A/B-type duplex structure. This is consistent with
our first study where both ON1-X and ON6-X displayed similar
CD-curves with RNA.9
In the case of the sulfonamide modification Z, the CD-curves
revealed somewhat different observations. For the DNA:DNA du-
plex, the single modification in ON1 demonstrated almost no
changes in the CD-spectrum, whereas the spectrum for ON2
demonstrated a lower band at 280 nm and a beginning shoulder
at 265 nm (Fig. 3). With the two modifications in ON3 and ON4,
this trend continued with the largest changes observed for ON3.
In other works, both the number of modifications and the se-
quence context influenced the gradual change in duplex struc-
ture. With the three modifications in ON5, an even larger band
at 265 nm was seen indicating the expected shift towards an
A/B-type duplex but also other changes in the CD curve were
seen. With ON6, the positive band has moved backwards to
275 nm, and the negative band at 250 nm has become very
small.
unmodified DNA:RNA duplex (Fig. 4). With the increasing number
of modifications, however, the intensity of the negative band at
250 nm was decreasing and moving towards 235 nm, and a new
small positive band appeared at 310 nm.
2.4. Molecular modelling
The DNA:RNA hybrid duplexes formed by the oligonucleotides
with two and four consecutive incorporations of monomer X, Y
14
and Z (ON3 and ON6) were built in MACROMODEL and studied
in molecular dynamics simulations. The initial hybrid structures
were built in the B-type duplex conformation and the incorpo-
rated monomers were subjected to a Monte Carlo conforma-
tional search verifying the C5 (pyrimidine)–C4 (triazole) bond
previously studied via ab initio calculations.9 The obtained low-
est energy structure was then subjected to a 5 ns molecular
dynamics simulation during which 500 structures were sampled.
These 500 structures were subsequently minimised, and the lo-
cal minimum structure obtained was used for further analysis.
Models of the resulting modified duplexes are shown in Figures
5 and 6.
The DNA:RNA duplexes with four consecutive incorporations of
either X,9 Y and Z (Fig. 5) were found to be A/B-type duplexes with
almost perfect stacking between both triazoles and phenyl moie-
ties. However, some differences were observed. In the duplex with
the phenol moieties Y, the aromatic rings seems to bend slightly
away towards the 50-end, perhaps from a repulsion between the
hydroxy groups. The duplex with the sulfonamide monomer Z,
on the other hand, demonstrated a perfect stacking of the aromatic
rings and apparently a consistent organisation between the neigh-
bouring sulfonamide groups. Furthermore, some distortion in the
duplex inclination was observed in all three cases but most pro-
nounced with Z. Thus, a short 2.8 Å distance consistent with hydro-
gen-bonding was observed between the second sulfonamide group
from the 50-end in ON6-Z and the 20-hydroxyl group of the 50-ter-
minal cytosine in the complementary RNA strand. This seemed to
be forcing the duplex to bend, although, the Watson–Crick base
pairing was conserved.
For the DNA:RNA duplexes containing the sulfonamide Z, all
CD-spectra indicated that the modified duplexes had an A/B-type
hybrid duplex conformation not differing significantly from the
Also the DNA:RNA duplexes with two consecutive incorpora-
tions of either X, Y and Z (Fig. 6) were found to be A/B-type du-
plexes. Stacking between the aromatic systems are observed in
Figure 1. CD spectra of the DNA:DNA duplexes containing one to three incorporations of X.

4706
N. K. Andersen et al. / Bioorg. Med. Chem. 18 (2010) 4702–4710
Figure 2. CD spectra of the DNA:RNA duplexes containing one to three incorporations of X.
Figure 3. CD spectra of the DNA:DNA duplexes containing one to four incorporations of Z.
all three cases, although the two modified nucleobases were tilted
out of the nucleobase plane pointing towards the 30-end of the
strand. This is less pronounced with the two monomers X but in
the 30-modification of the two, the co-planarity between the two
heterocycles appeared to be lost. For monomer Y, this tilting of
the modifications towards the 30-end was even more pronounced,
although no changes in the co-planarity were observed. An almost
similar structure is seen with monomer Z.
zoles, and click chemistry is hereby a convenient method for
introducing aromatic stacking in the major groove of nucleic acid
duplexes.
The hybridisation data for the oligonucleotides of the current
study followed the trend of our first study demonstrating that
one incorporation of
X lead to decreased stability of both
DNA:DNA and DNA:RNA duplexes, whereas four consecutive
incorporations of X demonstrate a net neutral effect on the sta-
bility of the DNA:DNA duplex and a massive increase in stability
of the DNA:RNA duplex. Herein, we have demonstrated that the
increase in duplex stability graduates by the number of modifi-
cations, and that two incorporations were enough to see the ma-
jor part of the effect. The reason for the significant decrease in
duplex stability obtained with single modifications with X might
be found in the hydrophobicity of the phenyl group and the
distortion in hydration of the duplex. This can, however, be
3. Discussion
In the present study we have demonstrated the simple and effi-
cient synthesis of two new nucleoside building blocks following
the CuAAC method. Clearly, 5-ethynyl-20-deoxyuridine, 1, consti-
tutes an obvious substrate for the easy preparation of various tria-

N. K. Andersen et al. / Bioorg. Med. Chem. 18 (2010) 4702–4710
4707
Figure 4. CD spectra of the DNA:RNA duplexes containing one to four incorporations of Z.
completely counterbalanced by the stacking of two phenyltriaz-
ole moieties as indicated by the very large increase in thermal
stability of DNA:RNA duplexes when going from ON1 and ON2
to ON3 and ON4. Also in the DNA:DNA duplexes some compen-
sation by the second modification was clearly seen. The third
and fourth consecutive incorporation of X lead to further relative
increase in stabilisation up to an increase in thermal stability of
the DNA:RNA duplex of 5 °C for each modification. The observa-
tion that only two consecutive incorporations of X were enough
to give significant duplex stabilisation is very important for prac-
tical RNA-targeting, as the number of possible target sequences
increases dramatically. Future studies will show, whether tria-
zoles attached to other nucleobases can demonstrate the same
duplex stabilisation by stacking with X hereby open the access
to even more potential target RNA-sequences.
the modified DNA:DNA, the increasing number of modifications
and hereby the increasing stacking in the major groove was fol-
lowed by a shift in duplex structure towards an A/B-type form as
demonstrated by CD-spectroscopy. In the DNA:RNA duplex, the
A/B-type was more or less retained when the modifications were
introduced.
With the specific goal of targeting RNA, oligonucleotides con-
taining enlarged bi- or tricyclic nucleobases or aromatic substitu-
ents on the nucleobases have been approached before.4,6 In the
case of the phenoxazine cytosine analogue, increased effect by
the number of consecutive incorporations due to stacking in
the major groove has also been demonstrated.4 Nevertheless,
the nucleoside monomers X, Y and Z from the current study
demonstrates that this stacking effect can be obtained with sim-
pler aromatic moieties obtained by straightforward click chemis-
The two new derivatives Y and Z displayed almost similar influ-
ence on duplex stability as X indicating only small influence from
the substituents on the distal phenyl position. In the DNA:DNA du-
plexes, the phenol Y seems to give the largest decreases in duplex
stability, whereas the partial compensation obtained with consec-
utive incorporations seems to be most pronounced with the sul-
fonamide Z. In the DNA:RNA duplexes, the only obvious
difference between X, Y and Z was that the stabilising effect of
stacking three or four building blocks is even more pronounced
with Z ending with the most stable duplex of the entire study with
a Tm of 55.5 °C corresponding to a gain in thermal stability of
+6.1 °C per modification.
When considering duplex structures as studied by modelling
and CD-spectroscopy, some differences between the three modifi-
cations were indicated supporting the hybridisation data. The sul-
fonamide modification Z seems to have the largest impact on
duplex structure of the three, probably due to the most efficient
stacking behaviour. The modelling data indicated some bending
of the DNA:RNA duplex with 4 ꢃ Z due to a hydrogen-bonding
interaction across the major groove, and the CD-spectrum sup-
ported some deviation from the standard A/B-type duplex.
try. This simple approach opens the possibility for a large
variation of different entities in the major groove and with a
much wider range of potential target sequences. The 5-(1,2,3-tri-
azole-4-yl)pyrimidine nucleosides can therefore be important fu-
ture building blocks for the development of antisense
oligonucleotides.
4. Conclusions
From the easily available 5-ethynyl-20-deoxyuridine, three sim-
ple nucleic acid building blocks introducing triazoles into the ma-
jor groove of nucleic acid duplexes have been obtained. The
stacking of triazoles, and the aromatic substituents attached, in
the major groove leads to very stable DNA:RNA duplexes—the
most stable containing a distal sulfonamide moiety. Efficient
RNA-targeting, and hereby therapeutic potential, can be obtained
with oligonucleotides containing only two consecutive incorpora-
tions of the triazoles.
5. Experimental section
The current study demonstrated the value of
p–p-stacking for
obtaining duplexes with increased thermal stability. The effect of
stacking was strongest in the DNA:RNA duplexes as compared to
DNA:DNA duplexes, which might be due to the A/B-type duplex
form being shorter and more compact than the B-type duplex. In
All commercial reagents were used as supplied. Reactions
were carried out under argon or nitrogen when anhydrous sol-
vents were used. Column chromatography was performed with
Silica Gel 60 (particle size 0.040–0.063 lm, Merck). NMR spectra

4708
N. K. Andersen et al. / Bioorg. Med. Chem. 18 (2010) 4702–4710
Figure 5. Modelling structures of modified DNA:RNA duplexes containing four consecutive incorporations of X, Y or Z. From left to right: ON6-X:RNA, ON6-Y:RNA, ON6-
Z:RNA. Blue: 5-substituents with the heteroatoms in light blue; red: backbone; green: nucleobases.
Figure 6. Modelling structures of modified DNA:RNA duplexes containing two consecutive incorporations of X, Y or Z. From left to right: ON3-X:RNA, ON3-Y:RNA, ON3-
Z:RNA. Blue: 5-substituents with the heteroatoms in light blue; red: backbone; green: nucleobases.
were recorded on a Varian Gemini 2000 spectrometer or a Bru-
ker Advance III 400 spectrometer. Values for d are in ppm rela-
tive to tetramethylsilane as an internal standard or 85% H3PO4 as
an external standard. Assignments of NMR-signals when given
are based on 2D spectra and follow standard nucleoside conven-
tion. ESI mass spectra as well as accurate mass determinations
were performed on a Thermo Finnigan TSQ 700 spectrometer.
Microwave heated reactions were performed on an Emrys™
Creator.
trated under reduced pressure. The residue was co-evaporated
with toluene (2 ꢃ 10 mL), and methanol (10 mL), and then purified
by column chromatography (0–10% CH3OH in CH2Cl2) to give the
nucleoside 5 (550 mg, 78%) as a white foam. Rf 0.3 (5% MeOH in
CH2Cl2). 1H NMR (DMSO-d6; 300 MHz) d 11.81 (s, 1H, NH), 8.72
(s, 1H, triazole-H), 8.40 (s, 1H, H-6), 7.77 (d, 2H, J = 8.9 Hz, Ar),
7.39 (m, 2H, Ar), 7.29–7.21 (m, 6H, Ar), 7.15 (m, 1H, Ar), 7.04 (d,
2H, J = 8.9 Hz, Ar), 6.82 (dd, 4H, J = 8.7, 1.5 Hz, Ar), 6.21 (t, 1H,
J = 6.3 Hz, H-10), 5.37 (d, 1H, J = 4.5 Hz, 30-OH), 4.23 (m, 1H, H-30),
3.98 (m, 1H, H-40), 3.68 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.25–
3.22 (m, 2H, H-50), 2.33–2.27 (m, 2H, H-20), 0.98 (s, 9H, (CH3)3C),
0.24 (s, 6H, (CH3)2Si). 13C NMR (DMSO-d6; 75 MHz) d 161.1 (C-4),
158.0, 155.3 (Ar), 149.7 (C-2), 149.4, 144.8 (Ar), 136.2, 135.5,
135.4 (C-6, Ar), 130.7 (C-4 triazole), 129.7, 129.6, 127.7, 127.6,
126.5, 121.9, 120.8 (Ar), 120.1 (C-5 triazole), 113.1 (Ar), 104.8 (C-
5), 85.7 (C-40), 85.7 (Ar3C), 85.3 (C-10), 70.4 (C-30), 63.6 (C-50),
54.9 (OCH3), 39.5 (C-20), 25.5 ((CH3)3C), 17.9 ((CH3)3C), ꢁ4.6
5.1. Synthesis of 5-(1-(4-(tert-butyldimethylsilyloxy)phenyl)-
1,2,3-triazol-4-yl)-50-(4,40-dimethoxytrityl)-20-deoxyuridine (5)
To a solution of nucleoside 4 (485 mg, 0.88 mmol) and the azide
10 in ethanol and water (10 mL, 7:3, v/v) was added CuI (108 mg,
0.57 mmol), sodium ascorbate (281 mg, 1.42 mmol) and pyridine
(3 mL). The mixture was stirred at rt for 4.5 h and then concen-

N. K. Andersen et al. / Bioorg. Med. Chem. 18 (2010) 4702–4710
4709
((CH3)2Si). HiRes ESI MS m/z (M+Na) found/calcd 826.3213/
826.3243.
(250 mg, 67%) as a white foam. Rf 0.5 (7.5% i-PrOH in CHCl3). 31P
NMR (CDCl3, 162 MHz) d 149.1, 148.7. HiRes ESI MS m/z (M+Na)
found/calcd 1030.3629/1030.3657.
5.2. Synthesis of 5-(1-(4-(N-((dimethylamino)methylidene)
aminosulfonyl)phenyl)-1,2,3-triazol-4-yl)-50-(4,40-dimetho-
xytrityl)-20-deoxyuridine (6)
5.5. Synthesis of tert-butyldimethylsilyl 4-azidophenylether (10)
A solution of the azide 9 (1.12 g, 8.27 mmol), tert-butyldimeth-
ylsilyl chloride (3.74 g, 24.8 mmol) and DMAP (0.5 g, 4.09 mmol) in
a mixture of pyridine and acetonitrile (32 mL, 1:1, v/v) was stirred
at rt for 48 h. The mixture was concentrated under reduced pres-
sure and the residue was co-evaporated with toluene (2 ꢃ 20 mL)
and methanol (10 mL). The residue was purified by column chro-
matography (CH2Cl2) to give the azide 10 (1.86 g, 90%) as a yellow
To a suspension of the nucleoside 4 (300 mg, 0.54 mmol), the
azide 12 (178 mg, 0.70 mmol), sodium ascorbate (65 mg,
0.32 mmol) and CuSO4ꢀ5H2O (25 mg, 0.1 mmol) in H2O/t-BuOH
(8 mL, 1:1, v/v) was added THF (1 mL) and pyridine (0.25 mL).
The resulting clear solution was stirred at rt for 14 h, and then di-
luted with CH2Cl2 (50 mL) and brine (30 mL). The phases were
separated, and the organic phase was washed with a saturated
aqueous solution of NaHCO3 (30 mL). The combined aqueous
phase was extracted with EtOAc (2 ꢃ 30 mL), and the combined
organic phase was dried (Na2SO4), and concentrated under re-
duced pressure. The residue was purified by column chromatog-
liquid. Rf 0.9 (CH2Cl2). IR (KBr) 2122.4 cmꢁ1 1H NMR (DMSO-d6,
.
300 MHz) d 7.01 (m, 2H, Ar), 6.89 (m, 2H, Ar), 0.94 (s, 9H,
(CH3)3C), 0.17 (s, 6H, (CH3)2Si). 13C NMR (DMSO-d6, 75 MHz) d
152.5, 132.3, 121.2, 120.2 (Ar), 25.5 ((CH3)3C), 17.9 ((CH3)3C),
ꢁ4.7 ((CH3)2Si). HiRes ESI MS m/z (M+Na) found/calcd 272.1184/
272.1190.
raphy (0–8% MeOH in CH2Cl2) to afford the nucleoside
6
(220 mg, 87%) as a white foam. Rf 0.3 (5% i-PrOH in CHCl3). 1H
NMR (CDCl3, 400 MHz) d 8.70 (br s, 1H, NH), 8.55 (br s, 1H,
HC@N), 8.17 (s, 1H, triazole-H), 8.06 (m, 2H, Ar), 7.90 (d, 2H,
J = 8.0 Hz, Ar), 7.40 (d, 2H, J = 7.6 Hz, Ar), 7.33–7.12 (m, 8H, Ar,
H-6), 6.82 (m, 4H, Ar), 6.32 (t, 1H, J = 6.0 Hz, H-10), 4.45 (br s,
1H, H-30), 4.07 (m, 1H, H-40), 3.74 (s, 6H, OCH3), 3.45–3.29 (m,
2H, H-50), 3.16 (s, 3H, CH3), 3.05 (s, 3H, CH3), 2.48 (m, 1H, H-
20), 2.31 (m, 1H, H-20). 13C NMR (CDCl3, 100 MHz) d 160.9,
158.58, 158.56, 144.6, 142.4, 135.7, 135.6, 130.1, 130.1, 128.3,
128.1, 127.9, 126.9, 120.2, 113.3, 86.9, 85.9, 77.2, 72.5, 63.6,
55.2, 41.6, 35.7. HiRes ESI MS m/z (M+Na) found/calcd
830.2610/830.2578.
5.6. Synthesis of N-dimethylaminomethylidene-4-azidobenzene-
sulfonamide (12)
To a cold stirred solution of POCl3 (0.98 mL, 10.6 mmol) in
dimethylformamide (20 mL) was added the azide 11 (1.05 g,
5.30 mmol). The reaction mixture was stirred at rt for 3 h, and then
poured into cold water and neutralised with saturated aqueous
ammonia. The formed precipitate was isolated, washed with water
(100 mL) and dried to afford the product 12 (0.98 g, 73%) as white
solid. Rf 0.4 (40% EtOAc in petroleum ether). Mp 155–156 °C. 1H
NMR (DMSO-d6, 400 MHz) d 8.21 (s, 1H, HC@N), 7.78 (d, 2H,
J = 8.4 Hz, Ar), 7.24 (d, 2H, J = 8.4 Hz, Ar), 3.14 (s, 3H, CH3), 2.90
(s, 3H, CH3). 13C NMR (DMSO-d6, 100 MHz) d 159.7, 142.8, 139.4,
127.8, 119.4 (Ar, C@N), 40.8, 35.0 (CH3).
5.3. Synthesis of 5-(1-(4-(tert-butyldimethylsilyloxy)phenyl)-
1,2,3-triazol-4-yl)-50-(4,4-dimethoxytrityl)-30-O-(P-(2-cyanoe-
thoxy)-N,N-diisopropylaminophosphinyl)-20-deoxyuridine (7)
5.7. Synthesis of oligodeoxynucleotides
The nucleoside 5 (213 mg, 0.26 mmol) was dried by the co-
evaporation with anhydrous CH2Cl2 (2 ꢃ 5 mL) and dissolved in
anhydrous CH2Cl2 (5.5 mL). DIPEA (0.23 mL, 1.33 mmol) and 2-cya-
Oligonucleotide synthesis was carried out on an automated
DNA synthesiser following the phosphoramidite approach. Synthe-
sis of oligonucleotides ON1–ON6 (X–Z) was performed on a
noethyl-N,N0-diisopropyl-phosphoramidochloridite
(177 lL,
0.79 mmol) were added, and the mixture was stirred at rt for 1 h,
15 min. The solution was diluted with CH2Cl2 (25 mL) and washed
with brine (25 mL) and water (25 mL). The organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by column chromatography (2–5% acetone in CH2Cl2)
to give the product 7 (196 mg, 74%) as a white foam. Rf 0.8 (10%
acetone in CH2Cl2). 31P NMR (DMSO-d6, 121.5 MHz) d 148.6,
148.3. HiRes ESI MS m/z (M+Na) found/calcd 1026.4308/
1026.4321).
0.2 lmol scale by using the amidites 3, 7 and 8 as well as the cor-
responding commercial 2-cyanoethyl phosphoramidites of the nat-
ural 20-deoxynucleosides. The synthesis followed the regular
protocol for the DNA synthesiser. For compound 3, 7 and 8, a pro-
longed coupling time of 20 min was used. 1H-Tetrazole was used
as the activator and coupling yields for all 2-cyanoethyl phospho-
ramidites were 95–99.8%. The 50-O-DMT-ON oligonucleotides were
removed from the solid support by treatment with concentrated
aqueous ammonia at 55 °C for 16 h, which also removed the pro-
tecting groups. The oligonucleotides were purified by reversed-
5.4. Synthesis of 5-(1-(4-(N-((dimethylamino)methylidene)
aminosulfonyl)phenyl)-1,2,3-triazol-4-yl)-50-(4,4-dimetho-
xytrityl)-30-O-(P-(2-cyanoethoxy)-N,N-diisopropylamino-
phosphinyl)-20-deoxyuridine (8)
phase HPLC on a Waters 600 system using a Xterra prep MS C18
10
;
l
m; 7.8 ꢃ 150 mm column; buffer A: 0,05 M triethyl ammo-
nium 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 20 min, quenched with
The nucleoside 6 (300 mg, 0.37 mmol) was dried by the co-
evaporation with anhydrous CH2Cl2 (2 ꢃ 10 mL) and dissolved in
anhydrous CH2Cl2 (10 mL). DIPEA (0.25 mL, 1.4 mmol) and 2-cya-
a aqueous solution of sodium acetate (3 M, 15
lL) and then added
noethyl-N,N0-diisopropyl-phosphoramidochloridite
(0.25 mL,
sodium perchlorate (5 M, 15 L) followed by acetone (1 mL). The
l
pure oligonucleotides precipitated overnight at ꢁ20 °C. After cen-
trifugation 12,000 rpm, 10 min at 4 °C, the supernatant was re-
moved and the pellet washed with cold acetone (2 ꢃ 1 mL) and
dried for 30 min under reduced pressure, and dissolved in pure
1.1 mmol) were added, and the reaction mixture was stirred at rt
for 4 h. The solution was diluted with CH2Cl2 (25 ml) and washed
with a 5% aqueous solution of NaHCO3 (2 ꢃ 10 mL). The aqueous
phase was extracted with CH2Cl2 (2 ꢃ 10 mL) and the combined or-
ganic phase was dried (Na2SO4) and concentrated under reduced
pressure. The residue was purified by column chromatography
(0–96% EtOAc in petroleum ether) to give the phosphoramidite 8
water (500 lL). The concentration was determined by UV at
260 nm, and the purity confirmed by IC analysis. MALDI-TOF-MS
[MꢁH]ꢁ gave the following results (calcd/found): ON2-X (2864.9/

4710
N. K. Andersen et al. / Bioorg. Med. Chem. 18 (2010) 4702–4710
2865.4); ON3-X (2994.0/2992.0); ON4-X (2994.0/ 2995.3); ON5-X
(3124.5/3123.9); ON1-Y (2880.9/2878.0); ON2-Y (2881.3/2876.5);
ON3-Y (3026.9/3024.7); ON4-Y (3026.0/3024.1); ON5-Y (3173.0/
3171.7); ON6-Y (3316.0/ 3315.8); ON1-Z (2943.0/2941.8); ON2-Z
(2943.0/2943.5); ON3-Z (3151.0/3146.0); ON4-Z (3151.0/)3147.0;
ON5-Z (3526.8/3522.4); ON6-Z (3567.0/3567.0).
300 K, time step 2.2 fs, SHAKE all bonds to hydrogen), during which
500 structures were sampled and subsequently minimised. The
duplex structures were minimised using the Polak–Ribiere Conju-
gate Gradient Method, the all-atom AMBER force field16,17 and GB/
SA solvation model18 as implemented in MACROMODEL V9.1. Non-
bonded interactions were treated with extended cut-offs (van der
Waals 8.0 Å and electrostatics 20.0 Å). Oscillator strength calcula-
tions for monomer Z were performed using a Hartree–Fock water
model with the aug-pc-1 basis set in GAUSSIAN 3.19
5.8. Thermal denaturation experiments
Extinction coefficients of the modified oligonucleotides were
estimated from a standard method but calibrated by the micromo-
lar extinction coefficients of the monomeric compounds X, Y and
Acknowledgements
dT, which were estimated from their UV-spectra (dT:
e
260 = 8.5,
The project was supported by The Danish Research Agency0s
Programme for Young Researchers, Nucleic Acid Center and The
Danish National Research Foundation, TheNucleic Acid Based Drug
Design Training Center (NAC-DRUG) in the Sixth Framework Pro-
gramme Marie Curie Host Fellowships for Early Stage Research
Training under contract no. MEST-CT-2004-504018, The Danish
Natural Science ResearchCouncil, Danish Center for Scientific Com-
puting and Møllerens Fond. Mrs. Birthe Haack and Mr. Christian
Schneider are thanked for technical assistance.
X: 260 = 7.8, Y: 260 = 12.6), and Z, which was estimated by ab ini-
e
e
tio calculated oscillator strength to be e260 ꢂ 8.0; practically we
used a e260 ꢂ 5.0. UV melting experiments were thereafter carried
out on a UV spectrometer. Samples were dissolved in a medium
salt buffer containing 2.5 mM Na2HPO4, 5 mM NaH2PO4, 100 mM
NaCl, and 0.1 mM EDTA at pH 7.0 with 1.5 lM concentrations of
the two complementary sequences. The increase in absorbance at
260 nm as a function of time was recorded while the temperature
was increased linearly from 5 to 60 or 75 °C at a rate of 0.5 or
1.0 °C/min by means of a Peltier temperature programmer. The
melting temperature was determined as the local maximum of
the first derivatives of the absorbance versus temperature curve.
The melting curves were found to be reversible. All determinations
are averages of at least duplicates within 0.5 °C.
References and notes
1. (a) Yan, H. Science 2004, 306, 2048; (b) Wengel, J. Org. Biomol. Chem. 2004, 2,
277.
2. Kurreck, J. Eur. J. Biochem. 2003, 270, 1628.
3. Luyten, I.; Herdewijn, P. Eur. J. Med. Chem. 1998, 33, 515.
4. Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873.
5. Wagner, R. W.; Matteucci, M. D.; Lewis, J. G.; Gutierrez, A. J.; Moulds, C. M.;
Froehler, B. C. Science 1993, 260, 1510.
5.9. Circular dichroism spectroscopy
6. (a) Gutierrez, A. J.; Matteucci, M. D.; Grant, D.; Matsumura, S.; Wagner, R. W.;
Froehler, B. C. Biochemistry 1997, 36, 743; (b) Gutierrez, A. J.; Terhorst, T. J.;
Matteucci, M. D.; Froehler, B. C. J. Am. Chem. Soc. 1994, 116, 5540; (c)
Sinkeldam, R. W.; Greco, N. J.; Tor, Y. ChemBioChem 2008, 9, 706.
7. (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004;
(b) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128.
8. (a) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057; (b)
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
CD-spectra were obtained at 5 °C using the same medium salt
buffer as in the UV melting experiments with 1.5
lM concentra-
tions of the two complementary sequences.
5.10. Molecular modelling
ˇ
9. Kocalka, P.; Andersen, N. K.; Jensen, F.; Nielsen, P. ChemBioChem 2007, 8, 2106.
The duplexes investigated (ON3-X–Z and ON6-X–Z) were built
with a standard B-type helical geometry within the MACROMODEL
V9.1 suite of programs.14 In all the calculations the phosphodiester
backbone charge was neutralised with sodium ions, placed 3.0 Å
from the negatively charged oxygen atoms in the plane described
by the phosphorus and the non-bridging oxygen atoms. The so-
dium–oxygen distances were restrained to 3.0 Å by a force con-
stant of 418 kJ/mol Å2. The modifications X, Y and Z were each
subjected to a Monte Carlo conformational search15 rotating the
C5 (nucleobase)—C4 (triazole) bond and the N1 (triazole)—C1
(phenyl) bond to generate 1000 structures, which were minimised
to identify the lowest energy structure. The lowest energy struc-
ture obtained from the Monte Carlo search was hereafter subjected
to a 5 ns molecular dynamics simulation (simulation temperature
10. (a) Burley, G. A.; Gierlich, J.; Mofid, M. R.; Nir, H.; Tal, S.; Eichen, Y.; Carell, T. J.
Am. Chem. Soc. 2006, 128, 1398; (b) Gierlich, J.; Burley, G. A.; Gramlich, P. M. E.;
Hammond, D. M.; Carell, T. Org. Lett. 2006, 8, 3639.
11. Buck, S. B.; Bradford, J.; Gee, K. R.; Agnew, B. J.; Clarke, S. T.; Salic, A.
BioTechniques 2008, 44, 927.
12. (a) Andersen, J.; Madsen, U.; Björkling, F.; Liang, X. Synlett 2005, 2209; (b) Cai,
Q.; Zhu, W.; Zhang, H.; Zhang, Y.; Ma, D. Synthesis 2005, 496.
13. Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Wullo, D.;
Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2007, 50, 1651.
14. MACROMODEL, Version 9.1, Schrödinger, LLC, New York, NY, 2005.
15. Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1989, 111, 4379.
16. Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.; Alagona, G.;
Profeta, S.; Weiner, P. J. Am. Chem. Soc. 1984, 106, 765.
17. Weiner, S. J.; Kollman, P. A.; Nguyen, D. T.; Case, D. A. J. Comput. Chem. 1986, 7, 230.
18. Still, W. C.; Tempcyk, A.; Hawley, R. C.; Hendrickson, T. J. Am. Chem. Soc. 1990,
112, 6127.
19. GAUSSIAN-03. Gaussian Inc., 340 Quinnipiac St., Bldg 40, Wallingford, CT 06492, USA.