A novel class of conformationally restricted oligonucleotide analogues: Synthesis of 2′,3′-bridged monomers and RNA-selective hybridisation

Article abstract of DOI:10.1039/a608231i

A novel 2′,3′-bicyclic nucleoside 5 has been synthesised and incorporated into oligonucleotide analogues resulting in strong and selective binding to an RNA complement.

Full text of DOI:10.1039/a608231i

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A novel class of conformationally restricted oligonucleotide analogues:
synthesis of 2A,3A-bridged monomers and RNA-selective hybridisation
Poul Nielsen,a Henrik M. Pfundhellera and Jesper Wengelb*†
a Department of Chemistry, Odense University, 5230 Odense M, Denmark
b Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark
A novel 2A,3A-bicyclic nucleoside 5 has been synthesised and
incorporated into oligonucleotide analogues resulting in
strong and selective binding to an RNA complement.
Six modified oligonucleotides were synthesised12 and evalu-
ated towards both complementary single-stranded DNA and
RNA.¶ As seen from Table 1, incorporation of one to four
modified bicyclic nucleotides X into a 14-mer destabilises
duplexes with the DNA complement dA14 by 2–3 °C per
modification, irrespective of X being incorporated in a row or
alternating with unmodified nucleotides. Analogously, a low-
ering of the melting temperature by approximately 3 °C per
modification was observed for duplexes with the RNA
complement rA14 when incorporating monomer X once or
several times alternating with unmodified nucleotides.
More encouraging results were obtained towards the RNA
complement when incorporating the bicyclic nucleoside X in a
row of four (5A-T5X4T5-3A), as a minor increase in melting
temperature compared to the unmodified control was observed.
Following this trend, an increase in the melting temperature of
Research towards modified oligonucleotides able to hybridise
with ssRNA (antisense or ribozyme approach) as a novel class
of therapeutic agents currently attracts much attention.1 The
potential of enhancing duplex stability using conformationally
restricted oligonucleotide analogues has been investigated, as
recently described in a review by Herdewijn.2 Bicyclo nucleo-
sides with an additional 3A,5A-ethylene bridge,3 bicyclic carbo-
cyclic nucleosides with a 1A,6A- or 4A,6A-methano bridge,4 2A,3A-
riboacetal
dimers,5
3A,6A-anhydrogluco-2A,5A-formacetal
trimers6 and conformationally restricted 1,5-anhydro-2,3-
dideoxy-d-arabino-hexitol nucleosides7 have been synthesised
and incorporated into oligodeoxynucleotides in order to obtain
an entropic advantage through the rigidity of the structures,
thereby increasing duplex stability. Here we report on the
synthesis and the promising melting results for 2A,3A-bridged
bicyclic pentofuranose oligonucleotides. This work was stimu-
lated by molecular modelling studies indicating the possibility
of a preferential 3A-endo conformation of the monomers as
required for RNA-selective binding.
ButMe2SiO
BnO
O
O
i–iii
O
O
O
O
O
OBn
Me
Me
Me
Me
The bicyclic nucleoside 5 was synthesised in twelve steps
from the known ulose 18 in an overall yield of 10% using a
strategy (see Scheme 1) where the additional alkyl chain was
introduced before, and the cyclisation accomplished after, the
nucleoside coupling step.‡ Whereas Grignard additions on
3A-keto nucleosides afforded xylo-configured isomers as major
products,9 the presence of the 1,2-O-isopropylidene group in
ulose 1 directs the addition of Grignard reagents to proceed
from the b-face of the furanose. Thus, the first step was a
stereoselective Grignard addition of an allyl group at the
3-C-position generating, after desilylation and concomitant
benzylation, the furanoside 2 with the preferred ribo configu-
ration in 59% yield. An allyl group was preferred to a vinyl
group as this Grignard addition was more successful in our
hands. The required inversion of the configuration at the
2-carbon could most conveniently be performed, using an
anhydro-approach,10 after the nucleoside coupling step. Using
standard methods, furanose 2 was deprotected and acetylated,
whereafter the 2A-O-unprotected nucleoside 3 was obtained in
79% yield (from 2) using the silyl Hilbert–Johnson/Birkofer
method as modified by Vorbru¨ggen and co-workers11 followed
by deacetylation. Mesylation and treatment with aqueous base
gave a 2-O,2A-O-anhydro intermediate which was directly
hydrolysed affording an arabino-configured nucleoside. The
double bond was oxidatively cleaved and 3A-C-hydroxyethyl
nucleoside 4 was obtained in 32% yield (from 3) after reduction.
Selective tosylation of the primary hydroxy group, cyclisation
using strong base and debenzylation afforded the nucleoside 5
in 68% yield (from 4).§ The new bicyclic nucleoside 5 was
incorporated into oligonucleotides as monomer X (see Scheme
1) after 4,4A-dimethoxytrityl(DMT)-protection of the
5A-hydroxy group and conversion to the corresponding phos-
phoramidite building block.
1
2
iv–vi
O
N
O
Me
Me
O
NH
NH
BnO
O
BnO
N
O
O
HO
vii–ix
HO
OBn
4
OBn OH
3
x–xii
O
N
O
Me
Me
NH
NH
O
N
O
O
HO
O
xiii–xv
O
O
O
OH
O
5
X
Scheme 1 Reagents and conditions: i, allylMgBr, Et2O, THF; ii, Bu4NF,
THF, 69% for two steps; iii, BnBr, NaH, DMF, 86%; iv, 80% AcOH, then
Ac2O, pyridine, 97%; v, thymine, N,O-bis(trimethylsilyl)acetamide,
MeCN, Me3SiOSO2CF3, 86%; vi, MeONa, MeOH, 95%; vii, MeSO2Cl,
pyridine, 89%; viii, NaOH, EtOH, H2O, 74%; ix, NaIO4, cat. OsO4, THF,
H2O, then NaBH4, THF, H2O, 49%; x, p-MeC6H4SO2Cl, pyridine; xi, NaH,
DMF, 83% for two steps; xii, H2, Pd(OH)2–C, EtOH, 82%; xiii, DMTCl,
pyridine, 97%; xiv, NC(CH2)2OP(Cl)N(Pri)2, EtN(Pri)2, CH2Cl2, 88%; xv,
DNA synthesiser
Chem. Commun., 1997
825

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12.5 °C was observed for the duplex between the (almost) fully
modified oligonucleotide 5A-X13T-3A and complementary RNA.
These results indicate that these new 2A,3A-bicyclo oligonucleo-
tides (‘2A,3A-BcNAs’) form a duplex structure with com-
plementary RNA which is thermally significantly more stable
than the corresponding unmodified DNA–RNA duplex. The
structure of the duplex 2A,3A-BcNA–RNA is probably different
from that of wild type DNA–RNA as indicated by the
destabilising effect of one or two bicyclic nucleosides in an
otherwise unmodified DNA strand. The shapes of the circular
dichroism spectra, known to be diagnostic of a particular duplex
structure, recorded for T14 :rA14 and X13T:rA14 are, however,
similar. It is thus clear that conclusions about the structural basis
for the observed increase in thermal stability must await results
from other sequences and from extensive molecular modelling
and NMR studies.
Degradation by nucleases signifies a serious limitation to in
vivo application of both natural and many modified oligo-
nucleotides. We have therefore evaluated the novel 2A,3A-BcNA
(X13T) against snake venom phosphodiesterase (a 3A-exo-
nuclease) using a method described earlier.13 Whereas the
unmodified control (T14) is fully degraded within 10 min, X13T
(or X13) remains intact for the 60 min monitored. Thus, although
other nucleases need to be evaluated, this result suggests that
2A,3A-BcNAs may be effectively protected against nucleolytic
degradation.
We thank The Danish Natural Science Research Council for
financial support. Dr Carl Erik Olsen is thanked for recording
MALDI-MS.
Footnotes
† E-mail: wengel@kiku.dk
‡ Full characterisation of all intermediates will be published as part of a
forthcoming publication.
§ The structure of nucleoside 5 was verified from MS, 1H NMR, 13C NMR,
1H–1H COSY and NOE experiments. Selected data for 5: 1H NMR (250
MHz; [2H6]DMSO): d 11.3 (1 H, br s, NH), 7.36 (1 H, d, J 1.1, 6-H), 5.80
(1 H, d, J 4.3, 1A-H), 5.61 (1 H, s, OH), 4.86 (1 H, m, 5A-Ha), 3.89 (1 H, d,
J 4.2, 2A-H), 3.85 (1 H, m, 2B-H), 3.83–3.64 (3 H, m, 4A-H, 5A-Hb, 2B-H),
2.14 (1 H, m, 1B-H), 1.81 (1 H, m, 1B-H), 1.78 (3 H, d, J 1.0, Me); 13C NMR
(62.9 MHz; CD3OD): d 166.7 (C-4), 152.2 (C-2), 139.7 (C-6), 110.1 (C-5),
89.4, 89.1, 85.5, 85.2 (C-1A, C-2A, C-3A, C-4A), 71.4 (C-2B), 61.6 (C-5A), 37.0
(C-1B), 12.7 (Me) (Found: C, 47.4; H, 5.7; N, 9.0; C12H16N2O6·H2O
requires C, 47.7; H, 6.0; N, 9.3%). Mutual NOE contacts between H-1A and
H-2A and between H-1A and H-4A, as well as the lack of contacts between H-5A
and H-1A or H-2A and between H-2A and H-6, confirm the assigned b-arabino
configuration.
¶ The oligonucleotides were synthesised on a Pharmacia Gene Assembler in
0.2 mmol scale using commercial 2-cyanoethyl phosphoramidites (2 min
coupling time, ca. 99% stepwise yield) and the amidite derived from
nucleoside 5 (2 3 12 min coupling time, ca. 95% stepwise yield).
Purification of 5A-O-DMT-ON oligonucleotides was accomplished using
disposable reversed-phase chromatography cartridges (Cruachem Inc.),
which includes 5A-O-detritylation. The composition of the oligonucleotides
were verified by MALDI-MS and the purity by capillary gel electro-
phoresis. Minor peaks ( < 5%) originating from X10T, X11T and X12T were
detected when running X13T.
In conclusion, a new bicyclic thymidine analogue 5 has been
synthesised and used as a monomer to construct a novel class of
conformationally restricted 2A,3A-bicyclo oligonucleotides
(‘2A,3A-BcNAs’). Melting experiments revealed excellent ther-
mal stability of a 2A,3A-BcNA–RNA duplex, which, together
with the observed stability towards 3A-exo nucleolytic degrada-
tion, suggests 2A,3A-BcNAs to be interesting candidates as
antisense molecules.
References
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Complimentary ssDNA
(dA14
Complimentary
ssRNA (rA14
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Oligonucleotide
Tm/°C
DTm/°C
Tm/°C
DTm/°C
5A-T14-3A
34.5
32.0
30.0
29.5
23.0
23.0
< 10
29.5
26.5
28.0
23.0
30.5
16.5
42.0
5A-T7XT6-3A
5A-T6X2T6-3A
5A-T6XTXT5-3A
5A-T5X4T5-3A
5A-T3(TX)4T3-3A
5A-X13T-3A
22.5
24.5
25.0
211.5
211.5
< 224
23.0
21.5
26.5
+1.0
213.0
+12.5
a Measured at 260 nm in medium salt buffer: 1 mm EDTA, 10 mm Na3PO4,
140 mm NaCl, pH 7.2; concentration of each strand: 1.0 mm; T = thymidine
monomer; dA = 2A-deoxyadenosine monomer; rA = adenosine monomer;
X = monomer derived from nucleoside 5; Tm = melting temperature
determined as the local maximum of the first derivative of the absorbance
vs. temperature curve; DTm = change in Tm compared to unmodified
controls.
Received in Glasgow, UK, 5th December 1996; Com.
6/08231I
826
Chem. Commun., 1997