DNA triplex structures are stabilized by the incorporation of 3'-endo blocked pyrimidine nucleosides in the hoogsteen strand

Article abstract of DOI:10.1016/S0960-894X(00)00434-0

A short route to pyrimidine locked nucleosides has been developed for their incorporation in triplex forming oligonucleotides (TFO). Compared to oligonucleotides built with standard nucleosides, the modified TFOs containing 3'-endo blocked residues formed

Full text of DOI:10.1016/S0960-894X(00)00434-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2287±2289
DNA Triplex Structures are Stabilized by the Incorporation of
30-endo Blocked Pyrimidine Nucleosides in the Hoogsteen Strand
Pascal Savy,a Rachid Benhida,a,* Jean-Louis Fourrey,a Rosalie Maurisseb
and Jian-Sheng Sunb
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 8 May 2000; revised 26 July 2000; accepted 2 August 2000
AbstractÐA short route to pyrimidine locked nucleosides has been developed for their incorporation in triplex forming oligo-
nucleotides (TFO). Compared to oligonucleotides build with standard nucleosides, the modi®ed TFOs containing 30-endo blocked
residues formed, with their corresponding DNA duplexes, more stable triple helix systems, an e€ect which might be ascribed to the
30-endo pucker of the modi®ed nucleoside residues. # 2000 Elsevier Science Ltd. All rights reserved.
At present, many advances are currently being made in
the understanding of the formation and the stabilization
of DNA triple helices which should help the develop-
ment of the potential therapeutic applications of triple
helix forming oligonucleotides (TFOs).1 Interestingly, it
has been shown that the stability of the pyrimidine
motif of triple helical structure is enhanced when the
TFO strand contains 20-O-methyl nucleosides. In this
case, the bene®cial stabilizing e€ect has been ascribed to
the 30-endo pucker of these nucleosides (N-type con-
formation).2 On the other hand, in a series of recent
papers, Wengel3 and others4 have shown that locked
nucleic acids (LNAs) incorporating 30-endo blocked
nucleosides form highly stabilized duplexes when
hybridized with their DNA complement. Similarly, it
was claimed that 20,50-oligonucleotides containing 20-
endo blocked nucleosides (S-type conformation) can
bind with a reasonable anity to their single-stranded
DNA target.5 These observations prompted us to
investigate whether such constrained nucleosides could
stabilize a pyrimidine-motif triple helix. To this aim, we
developed a new and short route to these nucleoside
derivatives starting from uridine (Scheme 1).
selectively tosylate the a-hydroxymethyl at the C-40
position. Despite many e€orts, we failed to ®nd condi-
tions which would provide the desired derivative in a
useful yield. Finally, the di-O-toluenesulfonyl derivative
2 was prepared in a modest 60% yield. Treatment of 2
with p-methoxybenzyl chloride a€orded the N-3 pro-
tected derivative 3 which, upon acidic removal of the
20,30-O-isopropylidene protection, provided quantita-
tively compound 4 as an immediate precursor of locked
nucleosides. Indeed, the treatment of 4 in the presence
of sodium hydride in dimethyl formamide (DMF) led to
the formation of a 1/4 mixture of the locked nucleoside
derivatives 5 and 6 in 90% combined yield,7 and these
were easily separated by ¯ash chromatography. They
were identi®ed by 1H NMR analysis and by comparison
of their free nucleosides (11 and 12) with those pre-
viously described (Scheme 2).3,4a
Subsequently, compounds 5 and 6 were separately
deprotected at the N-3 position by treatment with cerium
ammonium nitrate in the usual manner8 to give the 50-
O-toluenesulfonyl derivatives 7 and 8, respectively.
Removal of the 50-O-toluenesulfonyl group could not be
achieved by displacement with sodium hydroxide; however,
we were pleased to ®nd that the nucleophilic substitu-
tion took place readily using sodium benzoate in DMF
at 90 ꢀC to give the corresponding benzoates 9 and 10 in
high yield (95%). Their quantitative debenzoylation was
accomplished using sodium methylate in methanol to
a€ord the desired locked nucleosides 113 and 12.4a For
With derivative 1 in hand, readily obtained in two steps
from 20,30-O-isopropylideneuridine,6 we ®rst tried to
*Corresponding author. Tel.: +33-1-6982-3091; fax: +33-1-6907-7247;
e-mail: benhida@icsn.cnrs-gif.fr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00434-0

2288
P. Savy et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2287±2289
Scheme 1. Reaction conditions: (a) p-toluenesulfonyl chloride, DMAP, CH2Cl2, rt, 6 h, 60%; (b) p-methoxybenzyl chloride, K2CO3, DMF, rt, 3 h,
97%; (c) 80% acetic acid, re¯ux, 6 h, 100%; (d) NaH, DMF, rt, 1 h, 90% then chromatography separation (heptane:AcOEt: 1:1), 5:6 1:4.
Scheme 2. Reaction conditions: (a) cerium ammonium nitrate, acetonitrile/H2O, rt, 18 h, 95%; (b) sodium benzoate, DMF, 90 ꢀC, 3 h, 95%; (c)
NaOMe, methanol, rt, 10 min, 100%; (d) dimethoxytrityl chloride, pyridine, rt, 80%; (e) 2-cyanoethyl diisopropylchlorophosphoramidite, diiso-
propylethylamine, CH2Cl2, rt, 82%.
their ®nal incorporation into oligonucleotides II±III,
each nucleoside was ®rst 50-O-dimethoxytritylated and
the resulting derivative treated with 2-cyanoethyl diiso-
propylchlorophosphoramidite to give the corresponding
phosphoramidites 13 and 14 in good overall yields.9
probes I±II. DNA denaturation experiments were car-
ried out to assess the thermal stability of triple helices
formed by TFOs I±III in a 10 mM cacodylate bu€er at
pH 5.5 containing 100 mM NaCl, 10 mM MgCl2 and
0.5 mM spermine (Table 1). It was observed that the Tm
value of the triplex±duplex transition (T3m!2) of II is
30 ꢀC, whereas those of III and the unmodi®ed TFO (I)
are 5 and 24 ꢀC, respectively. Consequently, one can
Assays of triplex stability were made using a 29-mer
double-stranded target sequence using the 16-mer

P. Savy et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2287±2289
Table 1. The 29-mer ds DNA target was hybridized with its com-
2289
Acknowledgements
plementary third strand I±III (1.2 mM) in the indicated bu€er
Double-stranded 29-mer DNA target
We thank the `Association pour la Recherche contre le
Cancer' (ARC), the `Fondation pour la Recherche
Medicale' (FRM) and the MERT for ®nancial support
(to P.S. and R.M.). We are grateful to Dr. M. Thomas
for oligonucleotide syntheses.
30-CGTGAAAAA-TTTTCTTTTCCCCCCT-GACC-50
50-CCACTTTTT-AAAAGAAAAGGGGGGA-CTGG-30
Complementary third strands
I
50-TTTTCTTTTCCCCCCT-30
II
50-L1L1L1L1CTTTTCCCCCCT-30L1=11
III 50-L2L2L2L2CTTTTCCCCCCT-30L2=12
References and Notes
TFO
T3m!2 (ꢀC)a for
triplex±duplex
transition
1. Reviews: (a) Thuong, N. T.; Helene, C. Angew. Chem., Int.
Ed. Engl. 1993, 32, 666. (b) Frank-Kamenetskii, M. D.; Mir-
kin, S. M. Annu. Rev. Biochem. 1995, 64, 65. (c) Doronina, S.
O.; Behr, J.-P. Chem. Soc. Rev. 1997, 63. (d) Neidle, S. Anti-
Cancer Drug Design 1997, 12, 433. (e) Plum, G. E. Biopolymers
1997, 44, 241. (f) Gowers, D. M.; Fox, K. R. Nucleic Acids
Res. 1999, 27, 1569. (g) Praseuth, D.; Guieysse, A. L.; Helene,
C. Biochim. Biophys. Acta 1999, 1489, 181.
2. (a) Shimizu, M.; Konishi, A.; Shimada, Y.; Inoue, H.;
Ohtsuka, E. FEBS Lett. 1992, 302, 155. (b) Roberts, R. W.;
Crothers, D. M. Science 1992, 258, 1463. (c) Escude, C.; Sun,
J.-S.; Rougee, M.; Garestier, T.; Helene, C. C. R. Acad. Sci.
III 1992, 315, 521. (d) Ascensio, J. L.; Carr, R.; Brown, T.;
Lane, A. N. J. Am. Chem. Soc. 1999, 121, 11063.
3. Wengel, J. Acc. Chem. Res. 1999, 32, 301 and references
cited therein.
4. (a) Obika, S.; Morio, K.; Nanbu, D.; Imanishi, T. J. Chem.
Soc., Chem. Commun. 1997, 1643. (b) Obika, S.; Nanbu, D.;
Hari, Y.; Andoh, J.; Morio, K.; Doi, T.; Imanishi, T. Tetra-
hedron Lett. 1998, 39, 5401.
5. Obika, S.; Morio, K.; Hari, Y.; Imanishi, T. Bioorg. Med.
Chem. Lett. 1999, 9, 515.
6. Jones, G. H.; Taniguchi, M.; Tegg, D.; Mo€att, J. G. J.
Org. Chem. 1979, 44, 1309.
7. To date, we have not yet investigated the reaction condi-
tions which would favour one cyclization pathway over the
other (30 vs 20). An elegant solution to this problem might be
adapted from a recent work (Obika, S.; Hari, Y.; Morio, K.;
Imanishi, T. Tetrahedron Lett. 2000, 41, 215).
I
II
III
24
30
5
aT3m!2=melting temperature value (Æ1 ꢀC).
observe that incorporation of 40,20-locked nucleosides in
II provides 1.5 ꢀC stabilization per substitution as com-
pared to I.
This enhanced stability was expected since in 40,20-
locked nucleosides the sugar is constrained in C30-endo
conformation.3 In this respect, it is well known that
pyrimidine-motif triple helix formation is favoured by
20-substituents inducing C30-e0ndo conformation as
observed in the 20-methoxy or 2 -OH (RNA) series.2 In
contrast, a dramatic destabilization is observed with
40,30-locked nucleosides as present in III. This indicates
that, unlike to duplex DNA hybridization,5 such an
oligonucleotide, characterized by 20,50-phosphodiester
bonds and sugar moieties blocked in the C20-endo con-
formation, is unable to form stable enough triple helix
structures.
In conclusion, we have designed a new route to synthe-
size locked pyrimidine nucleosides 11 and 12 and we
have shown that the substitution of 20-deoxythymidine
by the corresponding uridine derived 40,20-locked
nucleoside residue 11 enhances the thermal stability in
the case of a pyrimidine-motif triple helix. It con®rms
also that the correct conformational pre-organization of
TFO favours the interaction between a TFO and its
target duplex sequence.10
8. Yoshimura, J.; Yamaura, M.; Suzuki, T.; Hashimoto, H.
Chem. Lett. 1983, 1001.
9. Oligonucleotides syntheses were performed on an Applied
Biosystem 392 synthesizer on a 1 mM scale using standard
synthesis cycle. After complete deprotection, oligonucleotides
were fully puri®ed by RP18 HPLC.
10. All intermediates and ®nal compounds were characterized
by their analytical and spectral data.