Binding properties of oligonucleotides containing a modified 2'-deoxyuridine with a thymine ended linker to pair with 2'-deoxyadenosine.

Article abstract of DOI:10.1016/S0960-894X(02)00062-8

Oligonucleotides containing in the place of thymidine the nucleoside 2, a 2'-deoxyuridine harbouring at C-5 a thymine ended linker, were found to undergo base pairing with the opposite 2'-deoxyadenosine. However, the corresponding duplexes are significantly destabilised as compared to the fully natural ones.

Full text of DOI:10.1016/S0960-894X(02)00062-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 981–983
Binding Properties of Oligonucleotides Containing a Modified
0
2
-Deoxyuridine with a Thymine Ended Linker to Pair with
0
2
-Deoxyadenosine
a
a
a,
b
Pascal Savy, Rachid Benhida, Jean-Louis Fourrey, * Rosalie Maurisse
b,
and Jian-Sheng Sun *
aInstitut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette, France
bLaboratoire de Biophysique, MNHN, 43 rue Cuvier, 75231 Paris Cedex 05, France
Received 26 October 2001; revised 10 December 2001; accepted 22 January 2002
0
Abstract—Oligonucleotides containing in the place of thymidine the nucleoside 2, a 2 -deoxyuridine harbouring at C-5 a thymine
0
ended linker, were found to undergo base pairing with the opposite 2 -deoxyadenosine. However, the corresponding duplexes are
significantly destabilised as compared to the fully natural ones. # 2002 Elsevier Science Ltd. All rights reserved.
The stability of DNA duplexes is controlled by an
intricate interplay of stabilising (hydrogen bonding to
form base pairs, base p-stacking) and destabilising
With this in mind, we proposed to consider another
approach, wondering if a pyrimidine nucleoside con-
struct featuring a linker bearing a terminal pyrimidine
could exercise a stabilising effect by forming with an
opposite adenosine, across the major groove, the unu-
(
electrostatic repulsion, steric effects) forces, not to
1
mention water molecule and metal ion interactions.
8
sual bonding outlined in Figure 1. We based our
2
With the ongoing developments of DNA technologies
assumption on the well known capacity of nucleic bases
to form Hoogsteen or reverse Hoogsteen hydrogen
bonds under a variety of conditions in the DNA as well
3
and oligonucleotide antisense applications, the need
has appeared to devise modified oligomers able to form,
with a complementary DNA or RNA strand, a duplex
with enhanced stability compared to a fully natural
9
as in the RNA series. Furthermore, we established, in a
previous work, that an oligonucleotide containing
internally the nucleoside 1 bearing the 4-thiothymine
photolabel was able to cross-link residues located in the
4
one. To date, many elegant proposals have been
worked out in order to modify the thermodynamics of
formation of nucleic acid complexes by influencing
either the enthalpic or the entropic contributing factors,
or both. In this regard, the most remarkable achieve-
ments in this field are probably the developments of
1
0
complementary strand under irradiation. This sug-
gested that, as least transiently, the photoactive base
could thread into the major groove of the duplex to
interact with the base acceptors of the opposite strand,
and this with a better efficiency for DNA than for RNA.
Moreover, this experiment suggested that the length of
the arm of compound 1 should be well designed for our
purpose.
5
peptide nucleic acids (PNA) and that of oligonucleo-
6
tides containing constrained nucleosides.
However, along this research line, other strategies have
been explored such as the one illustrated by the design
7
of G-clamp-containing oligonucleotides. A G-clamp
Herein, we describe our experiments aimed at the vali-
dation of the proposed concept. Nucleoside 2 was pre-
base corresponds to a cytosine analogue which can form
an additional hydrogen bond to guanine, contributing
to enhance considerably the helical thermal stability.
1
1
pared classically starting from intermediate 3 which in
1
2
the presence of ethylenediamine gave compound 4.
This derivative was conjugated with N-1-thyminyl-2-
1
3
acetic acid to provide intermediate 5 which was phos-
1
*
7
Corresponding author. Tel.: +33-1-6982-3053; fax: +33-1-6907-
247; e-mail: fourrey@icsn.cnrs.gif.fr (J. L. Fourrey); sun@mnhn.fr
4
phitylated to furnish phosphoramidite 6 (Scheme 1).
With this material in hand we could synthesise a series
(J.-S. Sun).
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00062-8

982
P. Savy et al. / Bioorg. Med. Chem. Lett. 12 (2002) 981–983
Figure 1.
Scheme 1. Reagents and conditions: (a) ethylene diamine, rt; (b) N-1-thymidinyl-2-acetic acid, EDC, HOBt, TEA, DMF, rt; (c) 2-cyanoethyl dii-
sopropylchlorophosphoramidite, N,N-disopropylethylamine, CH Cl , rt.
2
2
of five 10-mer oligonucleotides O-1, O-2, O-3, O-4 and
O-7 incorporating 1, 2, 3, 4 and 7 units of nucleoside 2,
respectively (Table 1). Their respective binding affinity
for their natural target 12AG was determined and com-
pared with that of the corresponding unmodified 10-mer
blocks of continuous modifications, are almost identical
underlining the non-cumulative substitution destabilising
effect.
1
5
As a consequence of these intriguing observations, we
searched to get insight into the nature of the eventual
interactions between the thymine moiety of 2 and its
corresponding opposed adenine residue. To this end we
proposed to investigate the behaviour of oligonucleo-
tides O-1 to O-7 in the presence of a modified target in
which four adenines have been replaced by N-7-deaza-
adenines (labelled a). N-7-Deazaadenine can partially
prevent Hoogsteen or reverse Hoogsteen hydrogen
bonding and thus be used as an internal probe. When
the corresponding 12-mer oligonucleotide designed
1
0-TC using UV melting experiments (Table 2).
Whatever the number and the position of nucleoside 2
within each oligonucleotide sequence, all the measured
melting temperatures (T ) are inferior to the one
m
observed with the reference duplex 12AG/10-TC. The
most salient feature which is worth notice is that there
is no linear variation between the Tm decrease and the
number of nucleoside 2 being incorporated in the
modified oligonucleotides O-1 to O-7. Thus, the sequen-
ces O-1 and O-3 with one and three modifications,
respectively, have the same affinity for their common
target 12AG. Comparing oligonucleotides O-2 and O-3
we find that the less modified sequence O-2 gives rise to
a less stable duplex than O-3. The two sequences differ
essentially by the mode of substitution which is dis-
continued in one case and continued in the other. The
12AaG was annealed with 10-TC the duplex was found
ꢀ
less stable than the reference duplex with a T of 25 C
m
ꢀ
ꢀ
corresponding to a 4 C decrease (1 C per substitution).
Accordingly, in the measurements of the stability of the
T s observed with O-4 and O-7, having one or two
m
Table 2. UV melting temperatures of duplexes between 12-mer and
1
0-mer sequences listed in Table 1
0
Sequence
12AG
29
12aAG
25
ÁTm
Table 1. Oligonucleotide sequences: a stands for N-7-deaza-2 -deoxy-
adenosine
1
0TC
ꢁ4
0
0
0
0
3
5
3
-TGAAAAGAAAAT-5
12AG
10TC
O-1
O-2
O-3
O-4
O-7
24
21
24
18
17
20
15
11
<5
<5
ꢁ4
ꢁ6
0
- CTTTTCTTTT-3
ꢁ13
0
-TGaAaAGaAaAT-5
12aAG
>ꢁ13
>ꢁ13
0
0
0
0
0
0
0
0
5
5
5
5
5
- CTT2TCTTTT-3
- CTT2TCTT2T-3
O-1
O-2
O-3
O-4
O-7
- CTTTTC222T-3
- C2222CTTTT-3
DNA thermal denaturation and renaturation experiments were carried
out in a 10mM cacodylate buffer (pH 6.6) containing 1 00 mM NaCl,
10mM MgCl2 and .05 mM spermine. Estimated accuracy of the
melting temperatures: ꢂ1 C.
0
0
- C2222C222T-3
ꢀ

P. Savy et al. / Bioorg. Med. Chem. Lett. 12 (2002) 981–983
983
duplexes involving oligonucleotides O-1 to O-7, this
decreased stabilty due to the presence of N-7-deazaade-
nine should be taken into account since it should be
observed whatever the nature of the base pair. Thus,
4. (a) Herdewijn, P. Liebigs Ann. 1996, 1337. (b) Herdewijn, P.
Biochem. Biophys. Acta 1999, 1489, 167.
5
. (a) Uhlman, E.; Peyman, A.; Breipohl, G.; Will, D. W.
Angew. Chem., Int. Ed. Engl. 1998, 37, 2797. (b) Nielsen, P.
Acc. Chem. Res. 1999, 32, 624.
6. (a) Wengel, J. Acc. Chem. Res. 1999, 32, 301. (b) Meld-
gaard, M.; Wengel, J. J. Chem. Soc., Perkin 1, 2000, 3539.
with sequences O-1 and O-2 the decreased ÁT found
m
ꢀ
ꢀ
to be 4 C and 6 C, respectively, is comparable to what
is observed with the unmodified target 12AG. Con-
versely, for the duplexes formed between 12AaG and
oligonucleotides O-3, O-4 and O-7 the variation of ÁTm
7
. Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-
Y.; Wagner, R. W.; Matteucci, M. D. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 3513.
ꢀ
ꢃ 13 C is higher than expected for a simple intrinsic
8. Our approach is to some extent reminiscent of the one
previously reported by Switzer who proposed to take advan-
tage of the p-stacking properties of adenine. See: Switzer, C.;
Prakash, T. P.; Ahn, Y. Bioorg. Med. Chem. Lett. 1996, 6, 815.
9. Blackburn, G. M.; Gait, M. J., Eds. Nucleic Acids in
Chemistry and Biology; IRL, Oxford, UK: 1990.
lost of stability due to N-7-deazaadenine replacement.
Considering all these results, we conclude that the pre-
sence of a bulky substituent (linker+thymine) at the C-
0
5
position of 2 -deoxyuridine 2 exerts a destabilising
effect on duplex formation. However, when nucleosides
are introduced blockwise the destabilisation is not
1
gaa, P. J. Chem. Soc., Chem. Commun. 1997, 167.
0. Saintome
´
, C.; Clivio, P.; Favre, A.; Fourrey, J.-L.; Lau-
2
cumulative as one could have expected. A reasonable
explanation can be suggested in the case of oligonu-
cleotides O-3, O-4 and O-7 with the contiguous thymine
moieties making favourable contacts, presumably by
means of p-stacking. Under these conditions, the con-
cerned residues might be pushed inside the major
groove of the double helix to escape hydrophobic inter-
actions and take advantage of hydrogen bonding with
the N-7 position of the complementary adenine.
1
1
1
1. Dreyer, G.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A.
985, 82, 968.
2. (a) Saintome
´
, C.; Clivio, P.; Fourrey, J.-L.; Woisard, A.;
, C.;
Clivio, P.; Fourrey, J.-L.; Woisard, A.; Laugaa, P.; Favre, A.
Favre, A. Tetrahedron Lett. 1994, 35, 873. (b) Saintome
´
Tetrahedron 2000, 56, 1197.
1
3. Compound 5: To a mixture of nucleoside 4 (340mg, 0. 5
mmol) and N-1-thyminyl-2-acetic acid (116 mg, 0.6 mmol) in
DMF (15 mL) were added succesively N-hydroxy-
benzotriazole (110mg, .07 mmol) and EDC (140mg, .07
mmol). After 3 h, the solvent was evaporated and the residue
purified by flash silica gel chromatography, elution with
CH Cl /MeOH (2–10% MeOH) containing 1% Et N gave
At best, this interpretation is tentative and more
experimentation will be required to characterise the
nature of the interactions that we have discovered with
nucleoside 2 and, in particular, to delineate the role of
the spacer arm. However, at this stage, it becomes clear
that we are dealing with two types of antagonising
forces suggesting that the entropic contribution of the
linker outbalances the enthalpic gain due to bonding
and stacking interactions which finally induces a con-
siderable destabilisation of the duplexes. Further
endeavour in this domain will be directed at the con-
struction of a less penalising linker.
2
2
3
nucleoside 5 as a white foam (374 mg, 87%). FAB-MS m/z 817
+
1
(M+ Li) . H NMR (300 MHz, CDCl ): d 7.62 (s, 1H, H-6),
3
7.44–7.21 (m, 10H, H-arom and H-6 T), 6.86 (d, J=8.7 Hz,
0
4H, H-arom), 6.29 (t, J=6.6 Hz, 1H, H-1 ), 4.46 (m, 1H, H-
0
0
3
6
), 4.01 (m, 1H, H-4 ), 3.77 (s, 6H, 2ꢄOMe), 3.39–3.23 (m,
0
H, 2ꢄH-5 and 2ꢄN-CH
2
), 2.44–2.09 (m, 4H, 2ꢄCH
2
), 1.94
(
s, 3H, Me).
4. Compound 6: To a solution of nucleoside 5 (85 mg, 0.1
Cl (5 mL) was added N,N-diisopropylethyl-
1
mmol) in CH
2
2
amine (0.4 mL) and cyanoethyl diisopropylchlorophos-
phoramidite (0.2 mL). The solution was stirred at room
temperature for 0.5 h, treated with an aqueous solution of
sodium bicarbonate (2x2mL), dried over magnesium sulfate
and evaporated under reduced pressure. The oily residue was
purified by column chromatography over silica gel with hep-
Acknowledgements
tane/EtOAc (1/1 to 2/8) containing 1% Et
3
N to give phos-
): d
We thank the ‘Association pour la Recherche contre le
Cancer’ (ARC), the ‘Fondation pour la Recherche
Medicale’ (FRM) and MERT for financial support (to
´
P.S. and R.S.). We are grateful to Dr. M. Thomas for
oligonucleotide syntheses.
phoramidite 6 (93 mg, 88%). 31P NMR (243 MHz, CDCl
3
1
1
37.6–137.3.
5. Standard oligonucleotides were purchased from Euro-
gentec and modified oligonucleotides were synthesised on an
0
Applied Biosystem 392 DNA/RNA synthetiser. The 5 -dime-
thoxytrityl (DMT) protected oligomers were purified by
reversed-phase HPLC (Waters PrepPak Cartridge Delta-Pak
C18, 15 mm, 300 A, 25ꢄ100 mm) using a 30 min linear gra-
dient of solvent A [0.1 M triethylammonium acetate buffer
References and Notes
(
pH 7) containing 7% of CH CN] and solvent B (CH CN)
3 3
(
100:0 to 60:40). The DMT protecting group was finally
1
. Kool, E. T. Annu. Rev. Biomol. Struct. 2001, 30, 1.
cleaved by treatment with 80% aq AcOH for 1 h at room
temperature. The oligomers were precipitated using an acetate
buffer (pH 6.2)/EtOH solution and isolated after centrifuga-
2
(
. (a) Seemann, N. C. Curr. Opin. Struct. Biol. 1996, 6, 519.
b) Seemann, N. C. Trends Biotechnol. 1999, 17, 437.
3
4
. Lebedeva, I.; Stein, C. A. Ann. Rev. Pharm. Toxicol. 2001,
1, 403.
ꢀ
tion at 0 C for 15 min.