Synthesis and DNA triplex formation of an oligonucleotide containing an urocanamide

Article abstract of DOI:10.1016/j.tetlet.2006.03.180

An urocanamide nucleoside designed and previously tested as its protected ribose derivative in aprotic solvents for binding a cytosine-guanine (CG) Watson-Crick base pair was successfully incorporated into a triplex forming oligonucleotide. Binding affini

Full text of DOI:10.1016/j.tetlet.2006.03.180

Tetrahedron Letters 47 (2006) 3849–3852
Synthesis and DNA triplex formation of an
oligonucleotide containing an urocanamide
Maria G. M. Purwantoꢀ and Klaus Weisz*
Institut fu¨r Chemie und Biochemie, Ernst-Moritz-Arndt-Universita¨t Greifswald, Soldmannstr. 16, D-17489 Greifswald, Germany
Received 27 February 2006; revised 23 March 2006; accepted 29 March 2006
Available online 27 April 2006
Abstract—An urocanamide nucleoside designed and previously tested as its protected ribose derivative in aprotic solvents for bind-
ing a cytosine–guanine (CG) Watson–Crick base pair was successfully incorporated into a triplex forming oligonucleotide. Binding
affinity and specificity of this nonnatural nucleoside were studied in a triple helix with duplex targets containing all four possible
Watson–Crick base pairs opposite the nucleoside analog in the third strand. UV melting experiments indicate the formation of a
well-defined triplex with specific binding of the urocanamide analog to a CG inversion of the homopurine–homopyrimidine target.
However, binding affinities in the triplex are weak and much lower when compared to the canonical base triads.
Ó 2006 Elsevier Ltd. All rights reserved.
As a carrier of genetic information, double-helical DNA
and its reliable sequence-specific recognition is central to
such basic life processes like replication and transcrip-
tion. Because the machinery of living cells ultimately
derives from genomic DNA, nucleic acids are attractive
targets for the artificial regulation of living systems.
Thus, manipulations through specific DNA binding
may block the expression of particular genes1,2 or result
in site-directed mutagenesis,3,4 but may also be used for
the mapping of genomic DNA.5,6 Various ligands have
been tested in the past for the sequence-specific recogni-
tion of double-helical DNA including duplex-invading
PNA,7,8 minor groove binding polyamides,9,10 and triple
helix forming oligonucleotides (TFOs).11,12 Pyrimidine
TFOs can bind in the major groove of a Watson–Crick
duplex by TÆA or C+ÆG Hoogsteen base pairing and are
particularly attractive with regard to their easy availabil-
ity and their potential to result in a nearly perfect struc-
tural fit upon binding to duplex DNA with its same
chemical subunits to form a triple helix. Unfortunately,
third strand oligonucleotides containing only natural
bases suffer from a limited recognition code and only
homopurineÆhomopyrimidine tracts within the duplex
are effectively recognized by the TFO. In order to over-
come these limitations, a large number of base analogs
have been developed for the binding to all four possible
base pairs in the past, however, success has been limited
mostly due to our restricted understanding of the inter-
play between the various interactions within such a
triple-helical system.13
Some time ago we have initiated studies aimed at getting
more detailed information on base–base interactions in
DNA triplexes. As a first step, we have designed and
synthesized novel nucleoside analogs based on urocanic
acid amides for CG Watson–Crick base pair recognition
and tested their hydrogen bond interactions with a free
CG pair in aprotic organic solvents.14,15 1H NMR
experiments in methylene chloride clearly indicated for-
mation of a base triple closely isomorphous to canonical
TÆAT and C+ÆGC triads within a regular pyrimidine or
parallel triplex motif and with the urocanamide specifi-
cally bound through two hydrogen bonds to the CG
base pair (Fig. 1). We now expand these model studies
at the monomeric level to the formation of triple-helical
complexes in an aqueous environment by incorporating
the N-alkylated urocanamide nucleoside U2 into the
TFO. To shed more light on the binding under aqueous
conditions and to compare with the results of our initial
model studies, third strand affinities towards all four
possible base pairs in the duplex opposite the novel base
analog in the TFO are determined through UV melting
experiments.
Keywords: Nucleoside analog; Triple helix; UV melting; Urocanamide.
*
Corresponding author. Tel.: +49 3834 864426; fax: +49 3834
864427; e-mail: weisz@uni-greifswald.de
ꢀ Present address: Faculty of Biotechnology, Universitas Surabaya,
Raya Kali Rungkut, 60292 Surabaya, Indonesia.
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.180

3850
M. G. M. Purwanto, K. Weisz / Tetrahedron Letters 47 (2006) 3849–3852
O
dRib
N
R
N
H
N
H
H
O
N
N
O
N
N
N
N
O
H
N H
N
N
N
N
H
H
N
O
H
H
dRib
dRib
N
N
N
N
dRib
dRib
H
H
Figure 1. Hydrogen bond mediated recognition of a CG base pair by an urocanamide receptor; arrows denote hydrogen bond acceptor and donor
sites, respectively.
The synthetic scheme for the preparation of the 50-trity-
lated and 30-phosphitylated 20-deoxyribonucleoside U2,
which was used as synthon for the oligonucleotide
synthesis, is outlined in Scheme 1. As described previ-
ously,14,15 N-alkylation of trans-urocanic acid 1 with
propylamine in the presence of N-hydroxysuccinimide
(NHS) and N-(3-dimethylaminopropyl)-N0-ethylcarbo-
diimide hydrochloride (DCI) afforded N-propyluro-
canamide 2. Glycosylation of 2 with a standard one-
3 with an anomeric a:b molar ratio of 1:2. The two ano-
mers were unambiguously assigned based on 2D NOE
intensities of sugar protons. After deprotection under
alkaline conditions followed by 50-tritylation, the b-ano-
mer 4 was separated from its a-isomer by HPLC (SiO2,
hexane/ethylacetate 10:1).18 After conversion to its
phosphoramidite, 5 was used as synthon for the synthe-
sis of the oligonucleotide OL15 using the standard
b-cyanoethyl phosphoramidite method. The 15-mer
oligonucleotide containing the nucleoside analog was
finally characterized by ESI mass spectrometry.
pot Vorbruggen procedure failed but could be achieved
¨
by employing the sodium salt of 2, which was prepared
in situ with sodium hydride and reacted with 2-deoxy-
3,5-di-O-p-toluoyl-a-D-ribofuranosyl chloride in aceto-
nitrile.16 The latter was prepared as previously de-
scribed17 with the a-anomer as the major product and
subjected to the reaction with the imidazole base with-
out further purification to afford the 20-deoxynucleoside
In order to examine the affinity and selectivity toward a
duplex target, equimolar amounts of four AT-rich
double-helical oligonucleotides were mixed with the U2
containing TFO OL15, which was designed to bind in
a parallel motif to the complementary duplex. Sequences
O
N
O
O
N
H
OH
N
HN
H
HN
N
N
N
ii
i
O
TolO
2
1
3
TolO
O
O
N
N
N
H
N
H
N
N
O
O
HO
HO
iv
N
iii
HO
U2
4
DMTO
O
Cl
P
CN
N
H
N
O
N
NC
O
O
O
P
(i Pr)2N
v
5
DMTO
Scheme 1. Reagents and conditions: (i) NHS, DCI, DMF, CH3CH2CH2NH2, 70%; (ii) 2-deoxy-3,5-di-O-p-toluoyl-a-D-ribofuranosyl chloride, NaH,
CH3CN, 37%; (iii) 1% NaOH in MeOH, 76%; (iv) DMTrCl, Et3N, pyridine, 40% (b-anomer); (v) i-Pr2EtN, CH2Cl2, 90%.

M. G. M. Purwanto, K. Weisz / Tetrahedron Letters 47 (2006) 3849–3852
3851
Table 1. Summary of triplex–duplex melting temperatures Tm (°C) of
triplexes with different ZÆXY base triplets
of the 15-mer double-helical targets differ in their
Watson–Crick base pair XY located opposite the
nonnatural U2 analog in a triplex and are given below:
ZÆXY base triplet
Tm (°C), pH 6.0
Tm (°C), pH 6.5
C+ÆGC
TÆAT
36.2
>39
26.5
21.0
21.7
29.2
35.8
21.9
20.2
18.4
OL15: 5’- C T
5’- G A A
3’- C T
T
C
G
C
U2 T
T
T
T
A
T
T
A
T
C T
G A A A A -3’
C T -5’
T T T -3’
GÆTA
X
Y
A A A
U2ÆCG
U2ÆTA
U2ÆGC
U2ÆAT
T
T
T
T
T T T
a
a
—
a
—
a
UV melting experiments at pH 6.0 of the 1:1 mixtures of
duplex and the TFO are shown in Figure 2. Each
mixture displays a sigmoidal high-temperature transi-
tion, which corresponds to the duplex to single strand
transition. In addition, hyperchromicity effects at lower
temperatures indicate the dissociation of the TFO from
the duplex. Note, however, that only the triplex with a
U2ÆCG base triad exhibits a sharp and cooperative
triplex–duplex transition at 21 °C whereas triplexes with
U2ÆAT and U2ÆGC triads only show a rather broad and
featureless, noncooperative transition at lower tempera-
tures. Compared to the U2ÆCG triplex, a cooperative yet
much broader low-temperature transition is observed in
case of the U2ÆTA triple-helical nucleic acid. These
results point to significant structural heterogeneity of
the U2ÆAT and U2ÆGC triplexes but to a well-defined
unique binding of the TFO in the U2ÆCG triplex.
—
—
a Transition not defined.
significantly hydrated and binding may therefore suffer
from a significant initial desolvation. Interestingly, a
similar situation with poor triplex stabilization was also
encountered for various base analogs carrying strongly
hydrophilic ureido substituents.20,21
Although designed for the selective hydrogen bond
mediated recognition of a CG base pair, the, albeit
broad, triplex–duplex transition of the U2ÆTA triplex
at a midpoint temperature of 22 °C at pH 6.0 also points
to some specific interactions between the U2 analog and
the TA base pair. Such an interaction might possibly
involve the formation of a hydrogen bond between a
protonated imidazole of U2 and the 4-carbonyl oxygen
of the thymine base as shown in Figure 3. Although
the free urocanamide is expected to be largely deproto-
nated at pH 6.0, the apparent imidazole pKa within a
U2ÆTA base triplet may be significantly elevated. In line
with the proposed base triplets, the triplex melting
temperature for the U2ÆTA triplex follows the well-
known pH dependence of the canonical triplexes with
protonated C+ÆGC triplets and decreases upon increas-
ing the pH to 6.5 (Table 1). However, an unprotonated
urocanamide binding a CG base pair should partially
counteract such a pH dependence and accordingly
triplex melting for the U2ÆCG triplex hardly changes
with increasing pH. Therefore, although still less stable,
the U2ÆCG triplex shows a relative stabilization with
increasing pH for the sequences tested.
However, U2 contributes only weakly to the stability of
a triplex with a CG inversion site. As summarized in
Table 1, melting temperatures are lower by P15 °C
when compared to triplexes containing only canonical
TÆAT and C+ÆGC triads and lower by 5 °C compared
to a noncanonical but moderately stabilizing GÆTA tri-
plex.19 Such a weak stabilization by the nucleoside ana-
log may be attributed to (i) the lack of a large surface
area necessary for optimal p stacking interactions and
(ii) the rather hydrophilic nature of the urocanamide.
Although we have increased the hydrophobicity of the
base by N-alkylation, the amide is still expected to be
.
U2 TA
.
U2 AT
2.5
2.4
2.3
2.2
2.1
2.0
.
U2 GC
In summary, our results on triplex formation with the
U2 containing TFO in aqueous solution is compatible
with the specific binding mode as predicted from our
model system of free nucleosides in aprotic solvents
and support a hydrogen-bonded U2ÆCG base triplet
closely isomorphous to the canonical base triads.
.
U2 CG
O
dRib
N
N
H
1.9
1.8
1.7
N
H
H
H3C
H
N
N
O
N
O
N
N
H
10
20
30
40
T(ºC)
50
60
70
dRib
N
N
dRib
Figure 2. Temperature-dependent absorbance at 260 nm of triplexes
with OL15. [Triplex] = 5.8 lM, 100 mM NaCl, 20 mM MgCl2,
pH = 6.0. Melting curves are offset for the sake of clarity.
Figure 3. Possible base triplet between analogue U2 and a TA
Watson–Crick base pair.

3852
M. G. M. Purwanto, K. Weisz / Tetrahedron Letters 47 (2006) 3849–3852
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´ `
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